Nanoscale apertures having islands of functionality

ABSTRACT

Methods, compositions and arrays for non-random loading of single analyte molecules into array structures are provided. Arrays of confined regions are produced wherein each confined region comprises a single island within the confined region. The island can be selectively functionalized with a coupling agent to couple a single molecule of interest within the confined region.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 13/095,021, filed Apr. 27, 2011, which i) claimspriority and benefit of Provisional Patent Application 61/329,026, filedApr. 28, 2010, and ii) is a Continuation-in-Part to U.S. patentapplication Ser. No. 12/384,097, filed Mar. 30, 2009, which claimspriority to and benefit of Provisional Patent Application 61/072,641,filed Mar. 31, 2008 and Provisional Patent Application 61/139,316, filedDec. 19, 2008; the full disclosures of which are incorporated byreference herein in their entirety.

BACKGROUND OF THE INVENTION

A variety of techniques in molecular biology and molecular medicine nowrely on analysis of single biological molecules. Such techniques includeDNA and RNA sequencing, polymorphism detection, the detection ofproteins of interest, the detection of protein-nucleic acid complexes,and many others. The high sensitivity, high throughput and low reagentcosts involved in single molecule analysis make this type of analysis anincreasingly attractive approach for a variety of detection and analysisproblems in molecular medicine, from low cost genomics to highsensitivity marker analysis.

For example, single molecule DNA sequencing is useful for the analysisof large sets of related DNAs, such as those that occur in a genome. Incertain of these methods, a polymerase reaction is isolated within anarray of extremely small (typically optically confined) observationvolumes that each permit observation of the enzymatic action ofindividual polymerases in each reaction/observation volume of the array,while the polymerase copies a template nucleic acid. Nucleotideincorporation events are individually detected, ultimately providing thesequence of the template molecule. This approach dramatically increasesthroughput of sequencing systems, and also dramatically reduces reagentconsumption costs—to the point where personalized genomics isincreasingly feasible.

The small observation volumes used for single molecule nucleic acidsequencing and other analysis methods are typically provided byimmobilizing or otherwise localizing the polymerase (or other) enzymewithin an optical confinement reaction/observation region, such as anarray of extremely smalls wells as in an array of Zero Mode Waveguides(ZMWs), and delivering a template, primers, etc., to the reactionregion. For a description of ZMW arrays and their application to singlemolecule analyses, and particularly to nucleic acid sequencing, see,e.g., “Selective aluminum passivation for targeted immobilization ofsingle DNA polymerase molecules in zero-mode waveguide nanostructures”(2008) Korlach et al. Proceedings of the National Academy of SciencesU.S.A. 105(4): 1176-1181; “Improved fabrication of zero-mode waveguidesfor single-molecule detection” (2008) Foquet et al. Journal of AppliedPhysics 103, 034301; “Zero-Mode Waveguides for Single-Molecule Analysisat High Concentrations” Levene et al. Science 299:682-686; publishedU.S. patent application No. 2003/0044781; Eid et al. (2008) “Real-TimeDNA Sequencing from Single Polymerase Molecules” Science DOI:10.1126/science.322.5905.1263b; and U.S. Pat. No. 6,917,726, each ofwhich is incorporated herein by reference in its entirety for allpurposes.

One difficulty in performing single molecule analyses occurs in loadingthe reaction/observation region of single molecule analysis devices withthe molecules of interest (e.g., template or other analyte and/orenzyme). Loading two or more molecules of interest into a ZMW or othersmall observation volume tends to complicate any analysis of signalsobserved from double (or more than double)-loaded region. This isbecause two (or more) sets of signals may simultaneously be observedfrom the ZMW or other observation volume, meaning that the signals fromthe ZMW would have to be deconvoluted before data from the observationregion could be used. More typically, data from double (+) loaded ZMWscan be recognized by various data analysis methods, and data frommis-loaded ZMWs or other relevant observation volumes is simplydiscarded.

To reduce the incidence of multiple molecule loading events in therelevant reaction/observation volume(s) of the array, it is typical inthe art to substantially “under-load” the array with the analytemolecules of interest. Random distribution of molecules into the arrayresults in one or fewer molecules being loaded into mostreaction/observation volumes when fewer than 37% of all observationvolumes are loaded. This type of loading is referred to as“Poisson-limited” analyte loading, meaning that few enough molecules areadded to the array so that a Poisson-style random statisticaldistribution of the analytes into the array results in one or feweranalytes per observation volume in most cases. In the ZMW context, stateof the art yields for single-molecule occupancies of approximately 30%have been obtained for a range of ZMW diameters (e.g., 70-100 nm). See,Foquet (2008), above. For this degree of loading, about 60% of the ZMWsin a typical ZMW array are not loaded (e.g., have no analyte molecules).

While such random distribution methods are effective in ensuring that,in most cases, not more than a single template or enzyme (or otheranalyte) molecule is loaded in each observation/reaction volume in anarray such as a ZMW array, it would be desirable to develop methods andcompositions for increasing the template and enzyme loading density ofsuch arrays. Higher loading densities would permit the simultaneousanalysis of more analyte molecules in the array, increasing thethroughput of such systems, while simultaneously decreasing analysiscosts. The present invention provides these and other features that willbe apparent upon complete review of the following.

SUMMARY OF THE INVENTION

In some aspects, the invention provides an array of nanoscale aperturescomprising: a transparent substrate having a cladding layer disposed onits surface, the cladding layer having a plurality of nanoscaleapertures extending therethrough; each nanoscale aperture having wallsand a base, wherein an isolation layer is on the walls and on a portionof the base of the nanoscale aperture; wherein a portion of the base ofthe nanoscale aperture comprises an island of substrate surrounded byisolation layer.

In some embodiments the substrate comprises a silica based material. Insome embodiments the substrate comprises a fused silica. In someembodiments the substrate comprises more than 10,000 nanoscaleapertures. In some embodiments the nanoscale apertures comprise holeshaving a circular lateral profile. In some embodiments the apertureshave a cross sectional dimension between 1 nm and 500 nm.

In some embodiments at least some of the plurality of nanoscaleapertures comprise a single molecule of interest. In some embodimentsthe single molecule of interest comprises an enzyme. In some embodimentsat least some of the plurality of nanoscale apertures comprise a singleactive polymerase enzyme, a single active template nucleic acid, or asingle active primer attached to the island.

In some aspects, the invention provides an array of nanoscale aperturescomprising: a transparent substrate having a cladding layer disposed onits surface, the cladding layer having a plurality of nanoscaleapertures extending therethrough; each nanoscale aperture having wallsand a base and each nanoscale aperture having an island of islandmaterial on the base of the aperture, wherein the island does notcontact the walls of the nanoscale aperture

In some embodiments the substrate comprises a transparent material. Insome embodiments the substrate comprises a silica based material. Insome embodiments wherein the substrate comprises a fused silica.

In some embodiments the island material comprises a metal. In someembodiments the island material comprises gold. In some embodiments thesubstrate comprises more than 10,000 nanoscale apertures. In someembodiments the nanoscale apertures comprise holes having a circularlateral profile. In some embodiments the apertures have a crosssectional dimension between 1 nm and 500 nm.

In some embodiments at least some of the plurality of nanoscaleapertures comprise a single molecule of interest. In some embodimentsthe single molecule of interest comprises an enzyme. In some embodimentsat least some of the plurality of nanoscale apertures comprise a singleactive polymerase enzyme, a single active template nucleic acid, or asingle active primer attached to the island.

In some aspects, the invention provides a method for producing an islandwithin nanoscale apertures on a substrate comprising: providing asubstrate having a cladding layer, the cladding layer having a pluralityof nanoscale apertures extending therethrough; conformally depositing asacrificial layer onto the top of the cladding layer; directionallyetching the sacrificial layer such that the sacrificial layer remains onthe walls of the nanoscale apertures, and the sacrificial layer isremoved from the region of the nanoscale aperture between thesacrificial layer on the walls; depositing an island material onto thesubstrate; etching the island material such that a portion of the islandmaterial remains in the region between the sacrificial layer between thewalls; and removing the sacrificial layer on the walls thereby producingan island of island material within the nanoscale aperture.

In some embodiments the method comprises providing a substrate having acladding layer, the cladding layer having a plurality of nanoscaleapertures; conformally depositing a first sacrificial layer onto the topof the cladding layer; directionally etching the sacrificial layer suchthat the sacrificial layer remains on the walls of the nanoscaleapertures, and the sacrificial layer is removed from the region of thenanoscale aperture between the sacrificial layer on the walls;directionally depositing an island material onto the substrate;depositing a second sacrificial layer on top of the island material;etching the second sacrificial layer so as to leave a portion of thesecond sacrificial layer on top of the island material within thenanoscale aperture, thereby protecting it from the subsequent etchingstep; etching the island material from the top of the cladding such thata portion of the island material remains in the region between thesacrificial layer between the walls; and removing the first sacrificiallayer on the walls and the second sacrificial layer, thereby producingan island of island material within the nanoscale aperture.

In some embodiments the substrate comprises a transparent material. Insome embodiments the substrate comprises a silica based material. Insome embodiments the substrate comprises a fused silica.

In some embodiments the island material comprises a metal. In someembodiments the island material comprises gold. In some embodiments thesacrificial layer comprises silicon or germanium or silicon andgermanium. In some embodiments the nanoscale apertures comprise holeshaving a circular lateral profile. In some embodiments the substratecomprises more than 10,000 nanoscale apertures.

In some embodiments the method further comprises binding a polymeraseenzyme, template nucleic acid, or primer selectively onto the island.

In some embodiments, the first sacrificial layer and the secondsacrificial layer comprise the same material. In some embodiments thefirst sacrificial layer and the second sacrificial layer comprisesilicon or germanium or silicon and germanium. In some embodiments thefirst sacrificial layer and the second sacrificial layer comprisesilicon.

In some aspects, the invention provides a method for forming an islandof substrate surface within a nanoscale aperture comprising: providing asubstrate having a cladding layer on top, the cladding layer having aplurality of nanoscale apertures extending therethrough; conformallydepositing an isolation layer on the cladding layer and exposed portionsof the substrate; conformally depositing a sacrificial layer onto thetop of the isolation layer; directionally etching the sacrificial layersuch that the sacrificial layer remains on the walls of the nanoscaleapertures, and the sacrificial layer is removed from the region of thenanoscale aperture between the sacrificial layer on the walls, exposinga portion of the isolation layer within the nanoscale aperture; etchingthe portion of the isolation layer within the nanoscale aperture toexpose a portion of the substrate; and removing the sacrificial layer toproduce a structure having an island of exposed substrate surfacesurrounded by isolation layer within each nanoscale aperture.

In some embodiments the isolation layer is deposited by atomic layerdeposition (ALD). In some embodiments the isolation layer comprises ametal oxide. In some embodiments the isolation layer comprises alumina.In some embodiments the substrate comprises a transparent material. Insome embodiments the substrate comprises a silica based material.

In some embodiments the substrate comprises a fused silica. In someembodiments the island material comprises a metal. In some embodimentsthe island material comprises gold. In some embodiments sacrificiallayer comprises silicon or germanium or silicon and germanium. In someembodiments the nanoscale apertures comprise holes having a circularlateral profile. In some embodiments the substrate comprises more than10,000 nanoscale apertures.

In some embodiments the method further comprises binding a polymeraseenzyme, template nucleic acid, or primer selectively onto the island.

In some aspects, the invention provides a method for forming an islandof substrate surface within a nanoscale aperture comprising: depositingan isolation layer onto a transparent substrate; forming nanopits in theisolation layer that extend to the substrate surface; depositing,exposing, and developing a resist to form a pillar of resist on top ofand extending over each nanopit; depositing a cladding layer such thatthe cladding layer covers the pillars of resist and the exposed regionsof isolation layer; and removing the resist resulting in lift-off of theportion of the cladding layer covering the pillars of resist, therebyforming a nanoscale apertures in the cladding layer, each having ananopit at its base surrounded by isolation layer.

In some embodiments the substrate comprises a silica based material. Insome embodiments the substrate comprises a fused silica. In someembodiments the isolation layer is deposited using atomic layerdeposition (ALD). In some embodiments the isolation layer comprisesalumina. In some embodiments the thickness of the isolation layer isbetween 2 nm and 20 nm. In some embodiments the nanopits have a circularlateral profile.

In some embodiments the nanopits have a lateral dimension of between 5nm and 40 nm. In some embodiments the nanoscale apertures have acircular lateral profile. In some embodiments the nanoscale apertureshave a lateral dimension of between 30 nm and 500 nm. In someembodiments the resist comprises a negative tone resist. In someembodiments the nanopits are formed using electron beam lithography orUV lithography.

In some embodiments the cladding comprises a metal. In some embodimentsthe cladding comprises aluminum.

In some embodiments, prior to forming the nanopits, alignment featuresare formed on the transparent substrate. In some embodiments thealignment features comprise features etched into the transparentsubstrate.

In some aspects, the invention provides a method for forming a nanoscaleaperture having an island of substrate surface within it comprising:providing a transparent substrate having a sacrificial layer on its top,the sacrificial layer having a hard mask layer on its top; patterningand etching the hard mask layer to form a plurality of nanoscalefeatures; etching the sacrificial layer to expose regions of thetransparent substrate such that the etch of the sacrificial layerextends underneath the hard mask layer, producing an undercut;conformally depositing the exposed portions of the transparent substrateand the tops of hard mask layer features with an isolation layer wherebythe isolation layer is deposited under the portions of the hard maskoverhanging the sacrificial layer; directionally depositing a claddinglayer, leaving portions of the isolation layer under the hard mask layerexposed; and removing the remaining portions of the sacrificial layer,hard mask, and portions of the isolation layer and cladding layer on topof the hard mask; thereby producing nanoscale apertures comprisingislands of transparent substrate within the nanoscale aperturessurrounded by isolation layer.

In some embodiments the sacrificial layer comprises silicon, germaniumor silicon-germanium. In some embodiments the hard mask comprises PECVDoxide or nitride.

In some embodiments the etching of the sacrificial layer is carried outin two etching steps, the first having substantially no undercut, andthe second etching step undercutting the hard mask layer.

In some embodiments the nanoscale aperture and the island within thenanoscale aperture each comprise a substantially circular lateralprofile. In some embodiments the nanoscale apertures have a lateraldimension between 30 nm and 500 nm. In some embodiments the islands havea lateral dimension between 2 nm and 40 nm.

In some embodiments the isolation layer comprises alumina. In someembodiments the cladding comprises a metal. In some embodiments thecladding comprises aluminum. In some embodiments the transparentsubstrate comprises a silica based substrate.

In some aspects, the invention provides a method for forming a nanoscaleaperture having an nanoscale island of island material within itcomprising: providing a stack of materials comprising from bottom totop, a transparent substrate, an island material layer, a sacrificiallayer, and a hard mask; patterning and etching the hard mask layer toform a plurality of nanoscale features; etching the sacrificial layer toexpose regions of the island material layer such that the etch of thesacrificial layer extends underneath the hard mask layer, producing anundercut; directionally depositing a cladding layer, leaving portions ofthe island material layer under the hard mask layer exposed; etching theexposed portions of the island material layer; and removing theremaining portions of the sacrificial layer, hard mask, and portions ofthe cladding layer on top of the hard mask; thereby producing nanoscaleapertures comprising islands of island material within the nanoscaleapertures surrounded by regions of transparent substrate surface.

In some embodiments the hard mask, and portions of the cladding layer ontop of the hard mask are removed before the etching of the exposedportions of the island material layer, and the removal of the remainingportions of the sacrificial layer is performed after this step.

In some aspects, the invention provides a method for forming a nanoscaleaperture having an nanoscale island of island material within itcomprising: providing a stack of materials comprising from bottom totop, a transparent substrate, a sacrificial layer, and a hard mask;patterning and etching the hard mask layer to form a plurality ofnanoscale features; etching the sacrificial layer to expose regions ofthe island material layer such that the etch of the sacrificial layerextends underneath the hard mask layer, producing an undercut;depositing an isolation layer whereby the isolation layer extends underthe undercut region to the remaining portions of sacrificial layer underthe undercut; directionally depositing a cladding layer, leavingportions of the isolation layer under the hard mask layer exposed; andremoving the remaining portions of the sacrificial layer, hard mask, andportions of the isolation layer and the cladding layer on top of thehard mask; thereby producing nanoscale apertures comprising islands ofsubstrate surface within the nanoscale apertures surrounded by regionsof isolation layer.

In some aspects, the invention provides a method of producing an arrayof nanoscale apertures wherein greater that 30 percent of the nanoscaleapertures comprises a single active molecule of interest comprising:producing an array of nanoscale apertures, the nanoscale apertureshaving walls and a base, the base having an island of island material,the island surrounded by isolation material; selectively binding themolecule of interest to the island whereby a fraction of the nanoscaleapertures comprise a single molecule of interest. In some embodimentsthe size of the island and the size of the molecule of interest areselected such that when one molecule of interest binds to the island, itsterically blocks the binding of a second molecule to the island.

In some embodiments the island has a substantially circular lateralprofile with a diameter of from about 2 nm to about 20 nm. In someembodiments the single molecule of interest comprises a single activemolecule of interest. In some embodiments the single molecule ofinterest comprises an enzyme. In some embodiments the single molecule ofinterest comprises a polymerase enzyme, a template nucleic acid, or aprimer. In some embodiments the island comprises material depositedwithin the nanoscale apertures.

In some embodiments the isolation material comprises the surface of thesubstrate. In some embodiments the island comprises a portion of thesurface of the substrate. In some embodiments the isolation materialcomprises aluminum oxide. In some embodiments the isolation materialcomprises a silane. In some embodiments the silane comprises asilane-polyethylene glycol (silane-PEG).

The invention provides methods and compositions for controlling loadingof single analyte molecules, such as nucleic acid templates, intoreaction/observation volumes (such as the wells of a ZMW array). Thesemethods and compositions are useful for increasing the throughput andefficiency of single molecule analysis systems. Basic approaches thatare provided include: creating a single binding site for an analyte inthe reaction or observation volume; removing excess binding sites viacatalytic or secondary binding methods, adjusting the size or charge ofthe analyte; packaging or binding the analyte molecules within (or on) aparticle, where a single such particle fits into the relevantobservation volume (due to size or charge of the particle and/orobservation volume); using non-diffusion limited loading; controllablyloading the analyte (e.g., using microfluidic or optical or electricalcontrol); sizing or selecting charges in the observation volumes (e.g.,the sizes of ZMWs in an array) to control which analytes will fit(spatially or electrostatically) into which array wells or well regions,iterative loading of analyte, e.g., by masking active sites betweenloading cycles, enriching the activity of the analytes that are loaded,using self-assembling nucleic acids to sterically control loading, usingribosome display to control loading and provide a base for analytescreening, adjusting the size of the reaction/observation volume; andothers. The methods and compositions provide for the possibility ofcompletely loading single molecule array reaction sites (instead ofabout 30% of such sites as occurs in the prior art using random “Poissonlimited” loading methods) with single analytes, and also provides forcontrol over size, charge and/or location features for both array wellsand analyte locations.

Accordingly, the invention provides methods of distributing a populationof molecules of interest or target molecules into a plurality of sizeconfined reaction or observation regions. Molecules of interest canoptionally comprise nucleic acids, proteins, and/or enzyme-substratecomplexes. The methods include providing a structure (e.g., a ZMW,planar substrate, small well array, or the like) comprising thesize-confined reaction or observation regions wherein each of theregions has an island of functional material and providing thepopulation of molecules of interest to be distributed into the confinedregions. The methods include adjusting the size of the confined reactionor observation regions by adding at least one sizing moiety toindividual reaction or observation regions, such that a selected numberof target molecules will fit into the resulting size-adjusted regions.Alternately, the size of individual target molecules of the populationcan be adjusted by linking at least one sizing moiety to individualtarget molecules, creating a population of sizing moiety-linked targetmolecules (e.g., particles linked to an analyte of interest). The sizingmoieties are of sufficient size, relative to the size-confined reactionor observation regions, so that only a selected number of sizingmoieties, e.g., less than 10 moieties, less than 5 moieties, or, e.g.,about one moiety, will fit into the size confined regions. The sizingmoieties can fit partly or fully into the region; the relevantdeterminant is delivery of the target molecule portion to the region.The methods thus include loading the target molecules into the regions,whereby a selected number of target molecules can fit into each region,thus distributing the population of target molecules into the pluralityof size confined regions. The methods optionally include selecting thesizing moiety or configuring the reaction region, so that a singlesizing moiety will fit into the reaction region. Optionally, the sizingmoieties or target molecules can comprise a selected charge, which canbe used to electrostatically control loading.

The size-confined regions can individually comprise or be present withinan individual well of an array, or in a size-delimited substrate, e.g.,a selected portion of a planar or other substrate. For example, thesize-confined regions can be present in an optically confined region,e.g., a reaction or observation region of a ZMW. Preferably, thepopulation of target molecules is distributed into size-confined regions(e.g., wells) of an array such that at least 38% of the size-confinedregions (e.g., wells) of the array are occupied by only one targetmolecule. For example, the population of target molecules can bedistributed into wells of the array such that at least 50%, or at least75% or more of the wells of the array are occupied by only one targetmolecule. Optionally, the methods include selecting the sizing moiety orconfiguring the reaction region, in such a manner that a single sizingmoiety will fit into the size-confined reaction region.

A sizing moiety is a moiety of a selected size that can be used toregulate entry of linked target molecules or linked island formingparticles into a size-confined region. Typically, the sizing moietiescan comprise one or more particles, e.g., beads, metal particles, ornanoparticles, or one or more polymers (e.g., one or more PEG,cross-linked polymers, dendritic polymers, hyperbranched polymers,starred polymers, dendrimers, dendrons, nucleic acids, DNA origami,polypeptides, or the like). In addition, sizing moieties can comprise apolysaccharide, polyethylene glycol (PEG), poly(lactic acid),poly(glycolic) acid, hyaluronic acid, a ribosome, a ribosomepolypeptide, or a type 1 collagen protein. In certain embodiments,sizing moieties can comprise viral capsids, e.g., viral capsids thatinclude at least one recombinant or modified coat protein that comprisespolymerase activity. In some embodiments, the sizing moities compriseparticles of island material surrounded by a coating, for example, by apolymer which can subsequently be removed to deposit an island of islandmaterial within the size-confined region. For example, the particle ofisland material can be a particle of metal or semiconductor that issurrounded by a polymeric coating so as to deposit one sizing moietyinto one size-confined region. The polymer can subsequently be degradedwith a plasma, resulting in the deposition of the island particle into acentral portion of the size-confined region.

The methods provided by the invention can be used to distributepolymerases to size-confined regions. In such embodiments, the sizingmoieties can comprise polymer tails linked to each polymerase, andprotease cleavage sites can be located between each polymer tail andpolymerase, e.g., to permit the release of the polymerase from thesizing moiety. Optionally, the sizing moieties can be ribosomes thateach bind a target polymerase during translation. A target population ofpolymerases in size-confined regions can optionally constitute aribosome display library of polymerase variants, such that differentpolymerase variants are present in different regions. Relatedly, themethods can further comprise screening the polymerases of the ribosomedisplay library for one or more properties of interest. The polymerasesof the library can optionally reverse transcribe or sequence a nucleicacid encoding the polymerase. This nucleic acid is at least initiallyassociated with a ribosome that is at least initially associated withthe polymerase.

In one embodiment, the sizing moieties expand upon binding to structuresin the confined regions to prevent additional sizing moieties fromentering into the confined regions. This can occur, e.g., where thesizing moiety is initially approximately spherical, and flattens uponentry into or binding within or proximal to the sizing region.Desirably, the sizing moieties and confined regions are sized such thata single sizing moiety can fit into each of the plurality of confinedregions, thereby providing for delivery of a single target molecule intothe size delimited region. As noted, individual sizing moieties can fitfully or only partially into each of the plurality of confined regionsto provide the target molecule (e.g., nucleic acid or protein), into theregion. In one convenient embodiment, the sizing moiety linked targetmolecules are flowed into the reaction/observation regions.

Sizing moieties can optionally form a size-exclusion matrix thatprevents more than a single target molecule from entering an analysis orfixation region of the size-confined reaction or observation region. Thefixation region can comprise, e.g., functionalized silicon, gold oraluminum; the functionalized region can comprise, e.g., one or morebinding partners; and the sizing moieties or analytes can comprise,e.g., one or more cognate binding partners. In one useful embodiment,the sizing moieties can be removed from the size confined reaction orobservation region subsequent to loading of the single target molecule.

Individual sizing moieties can be covalently or non-covalently linked towalls of the confined regions or to individual target molecules. Forexample, the target molecules to which the sizing moieties are linkedcan be polymerase enzyme molecules that comprise a reactive or bindingmoiety, such as a SNAP tag, that permits attachment of the sizingmoiety. Individual sizing moieties optionally can be cleaved from theindividual target molecules or walls after the loading step by exposingan individual sizing moiety-target molecule complex to, e.g., a changein pH, a change in salt conditions, addition of a competition moiety,light, heat, a protease, an endonuclease, an exonuclease, and/or anelectromagnetic field.

A plurality of size confined reaction or observation regions canoptionally include a subset of regions that are pre-loaded with a singlepolymerase molecule, a subset of regions members that lack polymerasemolecules, and sizing moiety-linked target molecules that comprise oneor more template nucleic acids. In such embodiments, the methods caninclude initiating copying or transcription of the template nucleic acidby the polymerase, followed by loading of additional polymerase proteinmolecules into at least some of the members that lacked polymerase,resulting in secondarily loaded confined observation or reaction regionscomprising secondary polymerase proteins. A secondary loading step canthen be performed in which additional sizing moiety linked templatenucleic acids are loaded into the secondarily loaded regions.

In a related aspect, the invention comprises methods of distributing apopulation of nucleic acid or other analyte molecules into a pluralityof wells in a small well array. The methods include providing a smallwell array that comprises the plurality of wells and providing apopulation of particles that bind or package a population of analytemolecules. In these methods, the plurality of wells in the array areindividually configured to receive a single particle from the populationof particles, such that delivering the population of particles into theplurality of wells distributes the population of analyte molecules tothe plurality of wells.

In one example, the invention provides methods of distributing apopulation of analyte molecules (e.g., nucleic acids, polymerasemolecules, etc.) to a plurality of wells in a zero-mode waveguide (ZMW).The methods include providing a zero-mode waveguide that comprises aplurality of wells, providing a population of particles that can bind orpackage a population of analyte molecules, and delivering the populationof particles to which the nucleic acids are bound or packaged into wellsof the ZMW. Optionally, the plurality of wells can be individuallyconfigured to each receive a single particle. Optionally, the particlescan be sized such that a single particle can fit in each of theplurality of wells.

In the embodiments, particles used to distribute nucleic acids or otheranalytes to the wells in a size delimited region, ZMW, or other arraycan optionally comprise viral capsids, e.g., capsids derived from alambda phage, a phi29 phage, a T7 phage, a T4 phage, a virus of theMyoviridae family, a virus of the Siphoviridae family, a virus of thePodoviridae family, or a capsid that comprises at least one recombinantcoat protein that comprises polymerase activity. Particles canoptionally comprise a self-assembled DNA structure. For example,self-assembled DNA structures used in the methods can optionallycomprise long DNAs, DNAs comprising a large radius of gyration,plasmids, circular DNAs, DNA origami structures, DNA grids, DNA gridscomprising a gold particles, DNA dodecahedrons, Sierpinski triangles,DNA octahedrons, or polycatenated DNA scaffolds. In certain embodiments,the DNA structure can comprise a single polymerase binding site and/orcan be covalently bound to a single polymerase molecule. Alternatively,the particles can individually comprise one or more nanostructure, bead,polymer, polysaccharide, polyethylene glycol (PEG), poly(lactic acid),poly(glycolic) acid, hyaluronic acid, type 1 collagen, ribosome,ribosome polypeptide, or polypeptide. Such particles can be cleaved fromthe nucleic acid molecules after delivery by exposing individualparticle-nucleic acid complexes to any one or more of the conditionsdescribed previously. These approaches can be used, for example todeliver a particle of island material into a well where the particle issmaller than the lateral dimensions of the well and is delivered in sucha manner that there is a region of exposed surface around the particle,such that the particle acts as an island.

Delivering the population of particles to e.g., to the wells of a ZMW ora small well array, includes distributing the particles such that at,e.g., least 38% of the wells, at least 50% or the wells, at least 75% ofthe wells, or, most preferably, at least 95% or more of the wells of theZMW or small well array are occupied by one particle. The methods canfurther include sequencing the nucleic acid molecules by performing asequencing reaction in the wells of the ZMW or small well array.

Compositions provided by the invention include analysis devicescomprising an array of analytes that are arranged in the array by one ormore phase determining features in such a manner that single moleculesof the analyte are present in each of at least 40% of the analysisregions of the array. The analyte molecules can optionally be, e.g., atleast 20 nm, at least 30 nm, at least 40 nm, or, preferably, at least 50nm apart on the array. The phase determining features that arrange theanalyte molecules can optionally include an arrangement of wells in thearray, an arrangement of ZMWs in the array, a mask that permits accessby the analyte to the analysis regions, an arrangement of particles inthe array, the particles comprising binding moieties that bind to theanalyte, and/or an arrangement of binding sites located at least 50 nmapart in the array, which binding sites are configured to bindindividual analyte molecules.

Other compositions that are provided by the invention include azero-mode waveguide (ZMW) or other small well array that comprises aplurality of wells, and a population of particles that bind or package apopulation of analyte that has been distributed into the plurality ofwells. Optionally, the wells of the ZMW or other array (or anobservation/reaction region in the ZMWs) can be configured to receiveonly one particle. Optionally, at least 38% of the wells, at 50% of thewells, at least 75% of the wells, or, most preferably, 95% or more ofthe wells of the ZMW or small well arrays of the invention can beoccupied by one particle. The particles in the wells can optionallycomprise one or more bead, nanostructure, or polypeptide, or viralcapsid recited above. The particles can be provided such that theparticles comprise both an island forming portion and a portion thatdoes not become part of the island, for example in the form of amicelle. The particles are provided to the wells in the controlled waydescribed herein, an the portion that does not become part of the islandis removed in order to deposit the particle within the well. In somecases the analyte such as the polymerase enzyme is present on the islandmaterial during deposition, in other cases, the analyte such as thepolymerase enzyme is attached to the island material after the particleis deposited into the well.

The invention also provides methods of producing a non-randomdistribution of single analyte molecules in analysis regions of anarray, e.g., analysis regions within wells of a small well array. Thesemethods include selectively distributing the analyte molecules into theanalysis regions, such that at least 38% of the regions are occupied byone analyte molecule, fewer than 5% of the analysis regions (and,preferably, fewer than 1%, or even fewer than 0.1%) are occupied by morethan one analyte molecule, and fewer than 62% of the analysis regionsare occupied by fewer than one analyte molecule. The non-randomdistribution of nucleic acid molecules in the analysis regions canoptionally be a non-Poisson distribution. In one useful embodiment,these methods can be used to distribute nucleic acid and/or polymerasemolecules to target wells of a zero-mode waveguide (ZMW). The nucleicacid molecules in the target wells can optionally be sequenced.

Non-random analyte molecule distribution to analysis regions in an arraycan optionally include configuring selected analysis regions of thearray to receive, at most, one particle, and delivering a population ofparticles that comprise, bind, or package the analyte molecules into thetarget regions, in a manner such that at least 38% of the regions areoccupied by the particles. The population of particles can optionally bedelivered to the analysis regions of the array such that at least 50%,at least 75%, or, most desirably, at least 95% or more of the analysisregions are occupied by the particles.

Producing a non-random distribution of analyte molecules in analysisregions of an array or ZMW can optionally include distributing a nucleicacid mask into individual analysis regions that comprise oligonucleotidepositioning features that position the nucleic acid mask within theindividual analysis regions. An individual analysis region can beexposed through a small hole in a selected region of the mask, theoligonucleotide positioning features can hybridize to the mask, and asingle analyte molecule can bind the analysis region through the smallhole in the mask. Optionally, the mask can be removed or degradedsubsequent to binding of the analyte molecule.

Optionally, the non-random distribution of analyte molecules in theanalysis regions can be produced by providing a population of nucleicacid particles individually comprising a single binding moiety andproviding a population of adaptors that can individually bind to thebinding moiety and to an individual the analysis region. Desirably, thenucleic acid particles are large enough relative to the analysis regionsto effectively inhibit binding of more than one particle to one analysisregion. Binding the population of nucleic acid particles and theadaptors to the analysis regions can be followed by the cleavage of thenucleic acid particles, which cleavage exposes individual singleadaptors bound to within the analysis regions. Analyte molecules canthen be advantageously bound to single adaptors.

Optionally, individual nanostructures comprising a binding site for theanalyte molecule can be fabricated in or distributed into the analysisregion in such a manner that binding of more than a single analytemolecule to the nanostructure is sterically inhibited. For example, ananostructure can optionally be a nanoparticle that is small enough toinhibit binding of more than a single analyte molecule comprising apolymerase to the nanoparticle. The nanostructure can optionally bedeposited electrochemically, and growth of the nanostructure can beterminated while the nanostructure is small enough to sterically inhibitbinding of more than a single analyte molecule to the nanoparticle.

Fabricating nanostructures in analysis regions can optionally compriseforming a monolayer of small nanoparticles in individual analysisregions and coalescing the small nanoparticles in the individual regionsinto larger nanoparticles in the regions, such that at least one largernanoparticle is formed in at least one individual region. Optionally, anarray of small wells comprising the analysis regions can be provided,and a micelle comprising a nanostructure of interest, which micelle issized such that it centers the nanostructure within the well, can bedistributed to each of the small wells. For example, the micelle cancomprise a coating of polymeric material having a small (island forming)nanoparticle in its core. After deposition of the micelles into thewells, the polymeric material can be removed to deposit the nanoparticleinto the well, for example in the central region of the well, thusforming an island of material in the well. The nanoparticle island thusformed can comprise an analysis region. The analyte can be specificallybound to the nanoparticle, attached either before or after thedeposition of the particle into the well. Alternately, fabricating thenanostructure in the region can include dispersing particles in aphotopolymerizable monomer, delivering the resulting monomer-particlesolution to the region, photopolymerizing the monomer in the region, andfixing the particle in the region.

Optionally, single nanostructure islands or nanostructure dots that binda single analyte molecule can be deposited into single analysis regionsof an array. The analysis regions of the array can optionally compriseregions proximal to the dots or islands, or they can comprise ZMWs thatare formed around the dots or islands. Optionally, an island or dot cancomprise Au—S—(CH₂)_(x)(C₂H₄O)_(y)-biotin, the analyte molecule cancomprises avidin-polymerase, and the analyte can be bound to the islandor dot through the binding of the Avidin moiety to the biotin moiety.Fabricating a nanostructure island or dot can include cleaning a fusedsilica or synthetic quartz wafer, applying a resist adhesion promoter tothe wafer, spin coating the wafer with a positive tone chemicallyamplified resist, baking the positive tone chemically amplified resist,performing e-beam lithography on the wafer to form a pattern in theresist, baking the resist after lithography, developing the resist,performing photoresist descum, depositing metal to form dots or islands,and deresisting the wafer.

A nanostructure island or dot is can optionally be fabricated in placeusing, e.g., electron beam lithography, nanoimprint pattern formation,high-aspect physical vapor deposition or chemical vapor deposition. Forexample, a substrate comprising a base material, a cladding material, anaspect buffer control layer, and a resist can be provided, an array ofwells, the wells extending through the resist, cladding material andaspect buffer control layer to the base layer can be formed, and amasking film over the array can be formed to produce a mask thatpartially extends across the tops of the wells of the array, restrictingaccess to a small diameter region in the bottom of each of the wells.Nanostructures in the small diameter regions can be deposited, and themask can subsequently be removed, thereby providing an array of wellsthat each comprise a single nanostructure that is configured to attach asingle analyte molecule in the well's analysis region. In otherembodiments, a substrate comprising a base material, a claddingmaterial, an aspect buffer control layer, and a resist can be provided,and an array of wells that extending through the resist, claddingmaterial and aspect buffer control layer to the base layer can beformed. A masking film can then be deposited over the array, therebyproducing a mask that partially extends across the tops of the wells ofthe array, restricting access to a small diameter region in the bottomof each of the wells. Subsequently, nanostructures can be deposited inthe small diameter regions, and the walls of the wells can be removed toprovide an array of nanostructure configured to attach a single analytemolecule in an analysis region of the array.

Alternatively, forming or depositing a nanostructure island ornanostructure dot that binds a single analyte molecule in an analysisregions of the array can include permitting an imperfect self-assembledmonolayer (SAM) to form in wells of a small well array or on the surfaceof a substrate. An island can then be formed through a selected regionof the SAM via atomic layer deposition. Other methods to form ananostructure in an analysis region include forming a multi-film stackon a substrate, forming a well array through multiple layers of themulti-film stack, depositing a spacer film over the well array,planarizing the multi-film stack to remove at least one layer of themulti-film stack between wells, and removing portions of the spacer filmwithin the wells, thereby producing nanostructures within the wells ofthe array. Optionally, a multi-film stack can be formed on a substrate,an array of structures can be formed through multiple layers of themulti-film stack, and a spacer film can be deposited over the array. Themulti-film stack can then be planarized to remove at least one layer ofthe multi-film stack, and the spacer film can be etched to producenanostructures on the substrate.

Methods to produce a non-random distribution of single analyte moleculesin analysis regions of an array can optionally include fabricating ananostructure array, wherein the analyte molecules are subsequentlybound to the nanostructures, and subsequently forming the analysisregions to encompass the nanostructures of the array. Fabricating thenanostructure array can optionally include forming an array of metalnanostructures on a substrate. For example, a cladding material can beapplied to the array, the cladding can be spin coated with a resistlayer, and regions of the resist proximal to the metal nanostructurescan be removed. The cladding in these regions can then be etched toexpose the metal nanostructures, thereby forming an array of small wellsin the cladding.

In other embodiments, single analyte molecules can be distributed toanalysis regions in a non-random manner by fabricating a small wellarray, wherein the floor of the wells comprises a substrate material andwalls of the wells comprise a cladding material that is different fromthe substrate material. The wells can then be coated with an analytebinding material, cladding material can be etched to increase thediameter of the wells, leaving the analyte binding materialapproximately in the center and on the bottom of individual wells in apatch of analyte binding material that is sufficiently small in size toinhibit binding of more than one analyte molecule to the patch ofbinding material. Analyte molecules can then be bound to the patch ofanalyte binding material in the wells.

Alternatively, a solvent comprising a low concentration of an analytebinding moiety that binds to the analyte and to analysis regions can bedeposited into an analysis region. The solvent can be evaporated todeposit the analyte binding moiety in the analysis region, and theanalyte can then be bound to the analyte moiety in the analysis regionto produce a non-random distribution of analyte molecules. In one usefulexample, the analysis region can be a zero mode waveguide (ZMW) andevaporation of the solvent can deposit the analyte binding moiety inapproximately the center of the ZMW.

Other methods of selectively distributing analyte molecules canoptionally include applying a coating in a solvent to the analysisregions, evaporating the solvent while rotating the array, therebyleaving a portion in the center of the analysis region that is free ofthe coating. A single analyte molecule can then be bound to the centerof the analysis region. Desirably, the uncoated center portion is smallenough that binding of more than 1 selected analyte molecule to thecenter region is sterically inhibited.

Single analyte molecule can optionally be controllably transported intoeach of the analysis regions to produce a non-random distribution ofanalyte molecules in analysis regions. For example, this can includefluidly coupling a plurality of analysis regions of the array to atleast one source of the analyte through at least one microscale channeland controlling the flow between the source and the analysis region witha control module that gates or regulates flow from the source to theanalysis region. A control module can optionally be operably connectedto a sensor configured to sense flow of an analyte molecules from thechannel into the analysis region. Optionally, the analyte can beoptically labeled, the sensor can comprise an optical sensor, and thecontroller can controls a valve between the source and the analysisregion. Optionally, the sensor can comprise a conductivity sensor thatdetects passage of an analyte molecule past the sensor, and each analytemolecule can be coupled to a dielectric nanoparticle. Single analytemolecules can optionally be transported into individual analysis regionsusing a gradient optical force, or an electrical trap. Controllablytransporting an analyte molecule into an analysis region can optionallypreventing the binding of additional analyte molecules in the analysisregions.

Optionally, selectively distributing single analyte molecules intoanalysis regions can include controllably transporting single thatcomprise a binding site for the analyte molecule into each of theanalysis regions. Steric inhibition can thereby prevent the binding ofmore than 1 analyte molecule to the particle. Alternately, the particlecan comprise a single analyte molecule binding site. A single particlecan be controllably transported into an analysis region via, e.g., afluidic control, an optical gradient, and/or an electrical trap. Anarray of optical traps with a trap to trap spacing that matches spacingbetween analysis regions can optionally be used to controllablytransport single to analysis regions. A plurality of single particlescan optionally be transported in parallel to a plurality of analysisregions.

Analyte molecules can optionally be activity enriched beforedistributing them into the analysis regions. For example, the analytemolecules can be or comprise polymerase molecules. Activity enrichmentof the polymerase can include binding polymerase molecules to a templatenucleic acid, separating unbound polymerase molecules from thetemplate-bound polymerases, thereby removing polymerase molecules thatlack template binding activity from polymerase molecules that comprisetemplate binding activity. The template bound polymerase molecules thatcan copy the template can also dissociate from the template, therebyforming released active polymerase molecules. (Polymerase molecules thatlack template copying activity remain bound to the template.)Alternately, activity enrichment of the polymerase molecules can includeremoving polymerase molecules that lack template binding activity frompolymerase molecules that comprise template binding activity, asdescribed above, and permitting template bound polymerase molecules tocopy the template. Based upon production of an at least partial copy ofthe template, the active polymerase molecules can be separated frominactive molecules. The actively enriched analyte molecules can beselectively reacted with the island or nanodot within a confined regionin order to attach a single active analyte molecule within the opticalconfinement.

The invention also provides a particle bound to a polymerase-templatecomplex. Any of the features noted above can apply to this embodiment,e.g., the particle can be a magnetic bead. For example, the magneticbead can include an affinity moiety such as a Ni-NTA moiety bound to thepolymerase template complex, e.g., where the polymerase comprises acognate affinity moiety such as a recombinant polyhistidine sequence.The polymerase can further comprises features that permit cleavage fromthe bead, such as a recombinant endonuclease site proximal to thepolyhistidine sequence.

In some cases, an array of single molecules of interest can be producedon a surface by having an array of nanodots on a transparent surfacewhere the array of nanodots is arranged such that each of the singlemolecules of interest can be observed independently. The nanodots are ofa size whereby only a single molecule of interest, e.g. an enzyme isbound to a single nanodot. The dots can be arranged into an array ofnanodots on the substrate by a variety of methods including using acore-shell polymer in which the core comprises the nanodot material or aprecursor to the nanodot material. Once the array of nanodots isdeposited, the nanodots can be selectively functionalized with acoupling agent for the molecule of interest. The nandots can then beexposed to the molecules of interest, for example where the nanodots andthe molecules of interest are sized such that where one molecule ofinterest binds to the nanodot, the binding of a second molecule ofinterest to that nanodot will be sterically prohibited. The array ofnanodots can be observed using a total internal reflection fluorescent(TIRF) system, or with a system wherein there are a plurality ofbeamlets, each directed to a nanodot on the array.

In some aspects the invention provides a method for forming an island offunctionality comprising: a) providing a substrate comprising a claddinglayer on top of a transparent layer, having a plurality of nanoscaleapertures extending through the cladding layer to the transparent layer,such that the apertures each comprise walls and a base; b) depositing acore-shell particle that is sized to fit within the plurality ofapertures onto the substrate such that generally only one core-shellparticle is deposited per aperture; c) removing the shell from thecore-shell particle whereby the core of the core-shell particle isdeposited on the base of the aperture; d) depositing an isolation layerover the substrate; and e) removing the core of the core-shell particleto produce an island of exposed substrate surrounded by isolation layer.

In some embodiments the invention further comprises, between steps (a)and (b), depositing a coating layer that covers both the cladding layerand exposed portions of the transparent layer, whereby after step (e) anisland of coating layer is exposed. In some embodiments, the core-shellparticle comprises a metal or metal oxide core and an outer organiclayer. In some embodiments, the invention further comprises, after step(e), selectively functionalizing the island of exposed substrate.

Combinations of these embodiments are expressly a feature of theinvention. Kits comprising the components noted herein are also afeature of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1H show various structures having islands of substrate (FIGS.1A-1D), or islands of island material (FIGS. 1E-1H) in confined regionswithin an array.

FIG. 2A is a schematic representation of a process of the invention forcoupling a single active molecule to an island of substrate within aconfined region. FIG. 2B shows the coupling of a single active moleculeto an island of island material within a confined region.

FIG. 3 is a schematic representation of a method for forming an islandof material within a confined region using two sacrificial layers.

FIG. 4 is a schematic illustration showing the formation of an island ofmaterial within a confined region using a sacrificial pillar.

FIG. 5A provides a photomicrograph of an array fabricated using a VistecVB300 Electron Beam System and positive-tone chemically-amplifiedresist. FIG. 5B shows a schematic and photomicrograph illustratingformation of a Ge nano-wire with a Au—Ge tip. FIG. 5C shows a flow chartand schematic for formation of a ZMW nanostructure over a nanodot array.

FIG. 6 provides a flow chart and schematic illustration of a process forimmobilization of nanoparticles in a ZMW.

FIG. 7A provides a schematic illustration of a procedure for formingnanoparticles in ZMWs. FIG. 7B provides a schematic of a process flowfor flat substrates.

FIG. 8A provides a schematic illustration of a process flow for formingnanoparticles in ZMWs via deposition of an imperfect monolayer. Aprocess flow for flat substrates is shown in FIG. 8B.

FIG. 9A provides a schematic illustration of a process for placing afunctionalized island in a ZMW. A related process flow for flatsubstrates is shown in FIG. 9B.

FIG. 10 shows a schematic process for immobilization of nanoparticles inZMWs.

FIG. 11 is a schematic illustration of a process for producing an islandof substrate material surrounded by isolation material within a confinedregion using a sacrificial layer.

FIG. 12 is a schematic illustration of an alternative process forproducing an island of substrate material surrounded by isolationmaterial within a confined region.

FIGS. 13A-13B show an example of a schematic for forming alignmentfeatures.

FIG. 14 shows a schematic of a method for producing an island ofsubstrate material using the deposition of a particle within a confinedregion.

FIG. 15 is a schematic illustration of a process for forming an islandof substrate surrounded by an isolation region within a confined regionusing a sacrificial pillar.

FIGS. 16A-16C show an example schematic of a chemical polishing processused to form an analyte binding site at the bottom of a ZMW or otherarray reaction region.

FIG. 17 provides a schematic of a tilted angle evaporation embodimentfor forming a functionalized region in a center of a ZMW.

FIG. 18 provides an example flow chart for enrichment of activepolymerases.

FIGS. 19A-19B provide additional details regarding polymeraseenrichment.

FIG. 20A shows a diagram outlining the dimensional elements for a methodof the invention for forming nanodot islands, FIG. 20B shows a diagramoutlining the dimensional elements for a method of the invention forforming islands of transparent substrate.

FIGS. 21A-21B show a transmission electron micrograph image of a crosssection of a ZMW having an island of substrate material.

FIG. 22 shows a transmission electron micrograph image of a crosssection of a ZMW having an island of exposed fused silica substrate atits center surrounded by an aluminum isolation layer.

FIG. 23 shows a transmission electron micrograph image of a crosssection of a ZMW having an island of exposed fused silica substrate atits center surrounded by an aluminum isolation layer.

FIG. 24 shows a transmission electron micrograph image of a crosssection of a ZMW having an island of exposed fused silica substrate atits center surrounded by an aluminum isolation layer including thedimensions of the island.

DETAILED DESCRIPTION

The invention provides methods and compositions that provide fornon-random distribution of target molecules (e.g., analytes such astemplate nucleic acids and/or relevant enzymes such as polymerases) intosmall reaction/observation volume arrays, such as ZMW arrays. Thesemethods and compositions can achieve much higher analyte, reagent,and/or reactant loading efficiencies than are typically observed usingPoisson-limited random molecule loading methods. The approachesgenerally involve creating a single binding site for the analyte in thereaction/observation volume, e.g., by placing or fabricating ananostructure in the reaction/observation volume, or by selectivelyforming analyte binding sites in the reaction/observation volume. Inaddition, the methods of the invention can include delivering theanalyte molecules using a sizing, e.g., particle, delivery system toprovide single molecule loading for each molecule type of interest.

These basic approaches can also be used in combination, e.g., the sizeand distribution of observation volumes can be selected in conjunctionwith a particle delivery system to control delivery and retention ofparticle-bound moieties of interest; binding sites can be used to bindto sizing moieties, iterative loading can be practiced in combinationwith any of the other approaches, etc.

The invention is generally directed to improving the loading of singlemolecule analytes into arrays of confined regions by providing an“island” within the confined region onto which the single analytemolecule can be attached. An island is a portion of the confined regionwhich has a different physical or chemical property from that of theportion of the confined region surrounding the island. Where theconfined region comprises walls, the island is generally separated fromthe walls. The island can be surrounded by what is referred to herein asan isolation region, or a region of isolation material. The isolationmaterial has one or more different physical and/or chemical propertiesfrom the island material. The different properties of the islandmaterial from the portion surrounding it within the confined region canbe used to selectively couple molecules of interest to the islandregion. The dimensions of the island material can be produced such thatonly a single molecule becomes attached to the island, for example wherethe first attached single molecule prevents the binding of a second. Bycontrolling the binding characteristics of the surfaces and the size ofthe island, the relative number of confined regions having only a singleanalyte molecule attached to them can be increased above the levels thatcan be obtained by random attachment.

The islands of the invention are generally on a substrate, which istypically a transparent substrate. In some cases, the island comprises alayer of material or a particle of material that is disposed on top ofthe substrate. In other cases, the island of material comprises aportion of the substrate that is not coated, but which is surrounded bya region of isolation material. In either case, the island can beselectively functionalized so as to be disposed to bind to a singleanalyte molecule of interest. In some cases, the island can comprise aparticle of island material that is deposited within the confinedregion. In this case, it is possible that the analyte molecule iscoupled to the particle prior to its being deposited onto the confinedregion.

FIG. 1 shows some embodiments of structures of the invention comprisingislands within confined regions which can comprise observation/reactionvolumes. Each of FIGS. 1(A) to (H) show a single nanoscale apertureextending through a cladding layer 110 to a transparent substrate 100.The observation/reaction volumes are generally present as an array ofobservation/reaction volumes, for example comprising hundreds tomillions of apertures on a substrate. The observation/reaction volumescan comprise, for example, zero mode waveguides. The substrate isgenerally transparent to allow for the illumination of the aperturesfrom below and to allow detection from below of light emitted from theapertures. The cladding is generally a thin layer of an opaque material,for example a metal or metal oxide. The cladding can have a thicknessfrom about 30 nm to about 200 nm. In FIGS. 1(A)-(D), each of theapertures has an island of exposed substrate 130 surrounded by isolationmaterial. The isolation material is a material that is different thanthat of the transparent substrate, allowing for selectivelyfunctionalizing the exposed transparent substrate. The transparentsubstrate can be selectively functionalized, for example using silanechemistry, to provide one or more coupling groups in order to attach ananalyte molecule of interest. In some cases, the array of confinedregions will include a layer of material between the transparentsubstrate 100 and the cladding 110. Where such a layer is present, theisland can comprise a portion of the exposed intermediate layer ratherthat an exposed portion of the substrate. The isolation material cancomprise an inorganic material, such as a metal oxide, e.g. alumina, orthe isolation material can comprise an organic material such as apolymer, e.g. polyethylene glycol.

FIG. 1(A) shows a nanoscale aperture having a layer of isolationmaterial that surrounds the island of substrate surface and also extendsover the walls of the aperture and the top surface of the claddinglayer. This type of structure can be advantageous in limiting the numberof types of surfaces which are to be functionalized. This type ofstructure can be formed using deposition and etching processes asdescribed in more detail herein. FIG. 1(B) shows an aperture in whichthe island of exposed surface is surrounded by isolation material on thebase of the aperture, and the isolation material does not substantiallyextend up the walls of the apertures or over the top of the cladding. InFIG. 1(C) the isolation layer 120 is disposed between the substrate andthe cladding. In FIG. 1(D) the isolation layer 120 surrounds the islandand extends over the walls of the aperture, but does not extend over thetop of the cladding.

FIGS. 1(E) to (H) show exemplary nanoscale apertures having islands 140of material disposed within them. The island material can be anysuitable material. The island material is generally different from thematerial comprising the substrate, allowing for selective coupling of ananalyte molecule of interest to the island. The nanoscale apertureextends through the cladding layer 110 to the transparent substratelayer 100. The island 140 is surrounded by a region 150, havingdifferent chemical or physical properties from the island material. Insome embodiments the island 140 comprises a metal, metal oxide, orsemiconductor material. In FIG. 1(E) the island 140 within the apertureis surrounded by regions 150 of exposed surface. In FIG. 1(F), theisland 140 comprises a particle 140 that has been deposited within theaperture leaving regions of the substrate 150 surrounding it within theaperture. This particle can be deposited with a method that results in alarge fraction of apertures having only one particle per well, forexample by depositing a particle having a core and a shell, where theparticle is sized such that generally only one particle is deposited ineach well, and removing the shell to deposit the core into the aperturesuch that the core of the particle comprises the island as used herein.FIG. 1(G) shows an aperture having an island that is deposited onto alayer 170 that is disposed on top of the transparent substrate 100,between the transparent substrate 100 and the cladding layer 110. Thelayer 170 can be a transparent material, or if it is thin, e.g. lessthan 10 nm, it can constitute a metal or semiconductor material whichwould not be transparent at higher thicknesses. The layer 170 can beused, for example, for access to different chemical properties thanthose of the transparent substrate surface. FIG. 1(H) shows an aperturein which a layer 170 of material surrounds the island 140, and extendsover the walls of the aperture and over the top of the cladding layer.As shown, the layer 170 does not extend under the island 140. In someembodiments, the layer 170 may extend under the island 140. The layer170 can comprise an inorganic or an organic material. It can comprise,for example, a metal, metal oxide, semiconductor, or polymer.

The structures shown in Figures (A) to (H) represent examples of islandstructures within confined regions. There are other structures that canbe used. It is understood that there are various combinations andvariations of the embodiments shown which are also of use The choice ofwhich of the structures or which combination of structures from FIGS.1(A) to (H) is used can depend on the effectiveness of the process usedto form the structures, the types of materials which are amenable to theprocess, and the performance of the fabricated array.

The confined regions generally have at least one lateral dimension onthe nanometer scale. The confined regions can comprise nanoscaleapertures with a lateral dimension between 20 nm and 300 nm, or between40 nm and 150 nm. The lateral dimensions of the islands are smaller thanthose of the nanoscale aperture such that the islands generally do notcome into contact with the walls of the apertures. In some cases thelateral dimensions of the islands are about 60% to about 2% or fromabout 40% to about 10% of the corresponding lateral dimension of thenanoscale aperture. In some embodiments the nanoscale aperture and theisland each have a substantially circular lateral profile. The nanoscaleaperture have a cylindrical aperture with a diameter between about 90 nmto about 150 nm, and the island can have a circular profile with adiameter of between about 5 nm and about 30 nm. The lateral profile ofthe apertures can be substantially circular, but need not be. In somecases, the lateral profile of the aperture is oval, elliptical,triangular, square, rectangular, polygonal, or in the form of a longstrip.

The arrays of confined regions comprising islands can be disposed on thetops of microarray structures for improved optical performance within ananalytical system. Such micromirror structures are described, forexample, in U.S. patent application Ser. No. 12/567,526, filed Sep. 25,2009, the disclosure of which is incorporated herein by reference forall purposes. In some cases, the confined regions have walls comprisinga layer of a non-reflective material as described in U.S. PatentApplication 61/241,700, filed Sep. 11, 2009, the disclosure of which isincorporated herein by reference for all purposes.

Having the analytical molecule of interest attached to the island canhave a number of advantages. One advantage being that only a smallnumber or only a single molecule or complex will be within the aperture.Another advantage is that the analyte molecule, when attached to theisland is held within the center of the aperture which can beadvantageous with respect to detection. For example, molecules disposedtoward the center of a ZMW can experience higher illuminationintensities than molecules right next to the wall of a ZMW. In addition,the emission from a molecule near the center of a ZMW can be transmittedmore effectively than molecules at the walls of a ZMW. In some cases,the attachment of the analyte molecule to a particle or nanostructurethat comprises the island can result in the enhancement of emission froma label associated with the analyte. This type of enhancement can beobtained, for example, where the material comprising the islandcomprises a metal. See, for example, U. S. Patent Application2008/0241866 filed Mar. 27, 2008. In some cases, this type ofenhancement is desired. In other cases, it is desired not to have thistype of enhancement, for example to enhance reproducibility. The methodsof the invention allow the user to produce a system with the level ofenhancement desired for the particular application.

The arrays and methods of the invention utilize arrays of nanoscaleapertures, such as ZMWs. These structures can comprise a transparentsubstrate upon which a cladding is deposited. The cladding has an arrayof nanoscale apertures extending through it to the transparentsubstrate. The cladding is generally from about 5 nm to about 300 nm, orabout 40 nm to about 150 nm thick. The cladding can comprise a metalsuch as aluminum, gold, chromium, copper, titanium, silver or platinum.

In the discussions herein, an “analyte” molecule or molecule of interestis a molecule analyzed in the system of interest, e.g., a templatenucleic acid, primer, enzyme, or the like. For example, in a sequencingreaction, the nucleic acid template can be an analyte molecule, as theproperties of the template (e.g., its sequence) are under investigation.However, the template is not the only analyte in a sequencing reaction.For example, properties of the sequencing primers are also detected inthe system (e.g., primer binding/polymerase initiation activity, asevidenced by a productive sequencing reaction) as are properties of theenzyme (e.g., polymerase activity, also as evidenced by a productivesequencing reaction). For convenience, unless context dictatesotherwise, the relevant analyte under consideration can be any moietyactive to the analysis, e.g., a substrate, template, primer, enzyme orthe like. Typically the analysis that is being performed is an opticalanalysis, and the analysis is performed by observing labels within theaperture. In some cases, the analysis includes observing the interactionof a bound species with another species in solution. In order to do so,the species in solution may be the species that is labeled, and theanalysis involves observing the label when the two species are boundtogether, forming a bound complex. Here, while the bound species may notbe labeled, we may still refer to it as a bound analyte as we areanalyzing its activity in observing its binding. In many cases, theanalyte comprises a complex such as an enzyme-template complex or anenzyme-template-primer complex or an enzyme-template-primer-substratecomplex. Where a complex is observed as part of the analysis, theattachment of any of the components of the complex comprises theattachment of an analyte.

FIG. 2 shows schematic illustrations of methods of the invention forattaching a single analyte molecule such as an enzyme within a nanoscaleaperture using either (A) an island of substrate 230 surrounded byisolation layer 220, or (B) an island of material surrounded bysubstrate surface or isolation layer. In FIG. 2(A) a nanoscale apertureextends through cladding 210 to substrate 200. An island 230 of exposedsubstrate surface within the aperture is surrounded by a layer ofisolation material 220. In step (IA) the substrate surface isselectively reacted with coupling agent 240. The coupling agent isselected to react primarily or exclusively with the substrate and to notreact with the isolation layer. In step (IIA) a single analyte molecule250 is attached to the island by reacting with the coupling agent 240. Ahigher than random distribution of single analyte molecules withinapertures can be obtained, for example where the size of the patch ofcoupling agent on the island is such that the binding of a first analytemolecule to the island sterically prevents the binding of a secondanalyte molecule. In some cases the analyte molecule comprises an enzymesuch as a polymerase enzyme. In some cases, the enzyme is provided inthe form of an enzyme complex, such as a polymerase-template orpolymerase-primer-template complex. In some cases, the analyte moleculesuch as the enzyme can have sizing moieties as described herein attachedto it to raise its effective size, to increase the steric prevention ofthe binding of a second analyte molecule. Subsequent or prior to thebinding of the molecule of interest, the isolation layer can be coatedwith a passivation layer to prevent inadvertent binding to that surface.The passivation layer can comprise polyethylene glycol.

In some cases, the transparent substrate 200 can comprise a silica basedmaterial such as fused silica, and the cladding material can comprise ametal layer such as aluminum. The isolation layer 220 can compriseeither an inorganic material such as alumina or an organic material suchas polyethylene glycol (PEG). The fused silica island can be selectivelyfunctionalized, for example using a silane coupling agent. The silanecoupling agent will react preferentially with the silica surface, andwill generally not react with the alumina or PEG surface. The analytemolecule is provides with a functionality that reacts specifically withthe coupling agent on the island. For example, in some cases, the silanecoupling agent comprises a silane-PEG-biotin, the analyte moleculecomprises an enzyme such as a polymerase comprising biotin, and theenzyme is bound to the island through an intermediate avidin orstreptavidin.

FIG. 2(B) shows an analogous process for binding a single analytemolecule 280 to an island of material 260 surrounded by substratesurface within the aperture. In step (IB) the island material isselectively reacted with coupling agent 270. The coupling agent isselected to react primarily or exclusively with the island material andto not react with the substrate surface. While shown here as unmodified,it will be understood that the surface of the substrate and the surfacesof the cladding will in some cases be treated with other layers in orderto modify their chemical or physical properties. For example, in somecases, one or both of these surfaces may be coated with PEG to preventunwanted binding. In step (IIB) a single analyte molecule is attached tothe island by reacting with the coupling agent 270. A higher than randomdistribution of single analyte molecules within apertures can beobtained, for example where the size of the patch of coupling agent onthe island is such that the binding of a first analyte molecule to theisland sterically prevents the binding of a second analyte molecule. Insome cases the analyte molecule comprises an enzyme such as a polymeraseenzyme. In some cases, the enzyme is provided in the form of an enzymecomplex, such as a polymerase-template or polymerase-primer-templatecomplex. In some cases, the analyte molecule such as the enzyme can havesizing moieties as described herein attached to it to raise itseffective size, to increase the steric prevention of the binding of asecond analyte molecule.

In some embodiments, for example, the island material comprises a metalsuch as gold. The gold can be selectively reacted with a coupling agent,for example using thiol terminated reagents such as athiol-alkane-PEG-biotin. The analyte molecule can comprise, for example,an enzyme such as a polymerase having attached to it a biotin moiety.The analyte can then be selectively reacted with the coupling agent onthe island with an intermediate avidin or streptavidin.

The selective immobilization illustrated in steps (IA) and (IB) of FIG.2 can be carried out as is known in the art by exploiting the chemicaldifferences between the material comprising the surface of the islandand the material surrounding the island. The differences in charge,polarity, hydrogen bonding, and reactivity of functional groups on thesurfaces can be used for obtaining selective immobilization. Approachesto selective immobilization are described, for example, in U.S. patentapplication Ser. No. 11/731,748 filed Mar. 29, 2007, the disclosure ofwhich is included herein by reference in its entirety for all purposes.

Particle/sizing moiety regulated delivery of analytes such as nucleicacids and/or enzymes such as polymerases to small volume arrays such asarrays of ZMWs can be accomplished by associating the nucleic acids orenzymes with the particles or other sized (and/or charged) moieties,e.g., by packaging the nucleic acids or enzymes using the particles(e.g., where the particles at issue comprise viral capsids), or bybinding or otherwise linking the nucleic acids or enzymes to theparticles. Any of a variety of particle types can be used, includingviral particles, proteins, protein complexes, beads, metallic particles,large molecules (e.g., PEG) and the like. Each of these approaches isdiscussed in more detail below.

Placing or Fabricating Analyte Binding Nanostructures into ArrayFeatures

One general approach of the invention to increasing the loadingefficiency of single molecule analytes into an array of reaction regionsincludes creating a single binding site for the analyte within each ofthe reaction regions, and then completely loading the single bindingsites. The single binding site can be, for example, an island ofmaterial deposited or formed within a confined region, or an island ofexposed substrate surface surrounded by an isolation region. Washingsteps can be used to remove unbound analytes from the array, resultingin essentially complete loading of analytes on the binding sites,leading to one analyte being loaded per reaction site. This yields anarray of reaction sites, such as an array of ZMWs, having most or all ofthe reaction regions of the array loaded with a single molecule of theanalyte of interest. While this approach is particularly well-suited toloading of single analyte molecules into reaction regions, it will beappreciated that the same approach can be used to load more than 1molecule, e.g., by creating more than one binding site per reactionregion, and loading the multiple binding sites.

In one example implementation, this aspect of the invention provides ageneral method for fabricating zero-mode waveguide (ZMW) or otherreaction region structures with a single nanostructure (e.g., a nanodot)inside the ZMW hole/reaction region or other array feature. The diameterof a typical ZMW hole is between, e.g., about 50 nm and about 120 nm,which is large enough to accommodate several copies of most reactionanalytes (polymerase molecules, templates, etc.). The nanostructure inthe hole or other reaction region, etc., is fabricated to be smallenough, relative to the analyte, that only a single molecule of theanalyte can bind to the nanostructure (alternately, the nanostructurecan include just a single binding site for the analyte). Examplenanostructures include islands comprising metal nanodots, metallicnanostructure, dielectric nanostructures, or semiconductor materialnanostructures that can be functionalized using standard chemistries todisplay binding moieties that can be bound by an analyte of interest.The nanostructures can also comprise islands having one chemicalcomposition surrounded by regions having another chemical composition,where the island is capable of being selectively functionalized for theattachment of a single molecule or single complex of interest.

For example, the presence of a functionalized metal nanodot or othernanostructure provides a binding site e.g., on the bottom surface orother target portion of a reaction region (e.g., a ZMW), etc., that issufficiently limited in area such that a single analyte (e.g.,polymerase or other enzyme) can be immobilized, e.g., for DNA sequencingor other single-molecule reactions. For example, noble metals, such asgold, silver, or platinum can be functionalized to form metal thiolatesusing alkanethiols, forming a binding site for the analyte.

Either the nanoparticles, the analytes, or both are optionallyfunctionalized in order to attach the analytes to the nanoparticles.Similarly, an intermediate binding moiety such as a biotin or avidin canbe functionalized. For instance, nucleotides or polypeptides herein areoptionally functionalized with alkanethiols to facilitate attachment tonoble metals such as gold. For example, nucleotides can befunctionalized at their 3′-termini or 5′-termini (e.g., to attach themto gold nanoparticles). See Whitesides, Proceedings of the Robert A.Welch Foundation 39th Conference On Chemical Research NanophaseChemistry, Houston, Tex., pages 109-121 (1995) and Mucic, et al. Chem.Commun., 1966, 555-557. Functionalization via alkanethiol attachmentstrategies is also optionally used to attach analytes to other metal,semiconductor or magnetic nanoparticles. Additional or alternatefunctional groups used in attaching analytes to nanoparticles caninclude, e.g., phosphorothioate groups (see, e.g., U.S. Pat. No.5,472,881), substituted alkylsiloxanes (see, e.g. Burwell, ChemicalTechnology, 1974, 4:370-377, Matteucci, J. Am. Chem. Soc., 1981,103:3185-3191 (1981), and Grabar, et al., Anal. Chem., 67:735-743).Nucleotides terminated with a 5′ thionucleoside or a 3′ thionucleosidecan be used for attaching nucleotides/oligonucleotides to solidnanoparticles. See also Nuzzo, et al., J. Am. Chem. Soc., 1987,109:2358; Allara, Langmuir, 1985, 1:45; Allara, Colloid Interface Sci.,1974, 49:410-421; Iler, The Chemistry Of Silica, Chapter 6, (Wiley1979); Timmons, J. Phys. Chem., 1965, 69:984-990; and Soriaga, J. Am.Chem. Soc., 1982, 104:3937. Further guidance regarding combinations ofnanoparticles and analytes can be found in, e.g., U.S. Pat. No.6,979,729 to Sperling et al.; U.S. Pat. No. 6,387,626 to Shi et al.; andU.S. Pat. No. 6,136,962 to Shi et al.; and U.S. Pat. No. 7,208,587 toMirkin et al. Additional details regarding suitable linking chemistriesis found herein.

One example of the overall strategy is to fabricate a nanostructurearray comprising Au nanostructures, followed by immobilization of apolymerase, template nucleic acid, or other analyte using typicalfunctionalization and binding chemistries, e.g., to provide an analytebound nanostructure, e.g.,AuS—(CH2)_(x)(CH₂H₄O)_(y)-Biotin-Avidin-Analyte (e.g.,AuS—(CH2)_(x)(CH₂H₄O)_(y)-Biotin-Avidin-Polymerase). The incorporationof metal nanostructures in ZMW holes or other reaction regions is notlimited by Poisson statistics; thus, binding of the polymerase or otheranalyte to the nanostructures provides high yields of reaction regionsin an array that each have a single active polymerase or other analyte.

Overall fabrication approaches to making the array of nanostructures inreaction regions optionally use available process technology fromsemiconductor fabrication, photomasking, and MEMS manufacturing. Forexample, an array of metal nanodots can be formed using e-beamlithography, Deep Ultra-Violet (DUV) lithography, nanoimprint, or otheravailable lithography process, or other available patterning techniques.Available commercial e-beam equipment and photoresist technology aresufficient to meet the size and positioning resolution requirements,e.g. a Vistec VB300 Electron Beam System and positive-tonechemically-amplified resist. The steps can include, e.g.: (1.) surfacecleaning of a fused silica or synthetic quartz wafer, e.g., usingconventional industry standard RCA protocols (also known as “standardcleaning” or SC-1), or using Piranha cleaning (also known as “piranhaetching,” e.g., using a mixture of sulfuric acid and hydrogen peroxide),see also, Rastegar “Cleaning of Clean Quartz Plates,” Surfacepreparation and Wafer Cleaning Workshop, Austin, April 2005; (2)application of a resist adhesion promoter such as, but not limited to,hexamethyldisilazane; (3) spin coating and post-application bake of apositive-tone, chemically-amplified resist; (4) e-beam lithography; (5)post-exposure bake; (6) photoresist development; (7) photoresist descum;(8) metal deposition; and (9) deresisting. For additional details inwafer fabrication and lithography, see, e.g., Eynon and Wu (2005)Photomask Fabrication Technology, New York, McGraw-Hill; Alexe (Editor),Gösele (Editor), Gösele (Author) (2004) Wafer Bonding Springer ISBN-10:3540210490; Luo (2004) Integrated Modeling of Chemical MechanicalPlanarization for Sub-Micron IC Fabrication: from Particle Scale toFeature, Die and Wafer Scales ISBN-10: 354022369X; Madou (2002)Fundamentals of Microfabrication: The Science of Miniaturization, SecondEdition CRC; and Atherton (1995) Wafer Fabrication: Factory Performanceand Analysis (The Springer International Series in Engineering andComputer Science) Springer ISBN-10: 0792396197. Microfabricationapproaches which can be used to form the structures described herein aredescribed in copending U.S. patent application Ser. No. 12/567,526 filedSep. 25, 2009, and 61/312,953 filed Mar. 11, 2010, each of which areincorporated herein by reference in their entirety for all purposes.

If metals such as Au, which generally have poor adhesion to SiO, areused, then an adhesion promoter can be deposited using vapor phasedeposition. One example for an adhesion promoter for Au on SiO isoctadecyltrichlorosilane (Szunerits et al. (2006) 22:10716-10722). Otheralternatives to improve adhesion include using an interfacial metal suchas Cr or Ti during the metallization step. Further details regardingavailable deposition methods, including vapor and thin film deposition,can be found, e.g., in Harsha (2006) Principles of Vapor Deposition ofThin Films, Elsevier Science ISBN-10: 008044699X; Dobkin and Zuraw(2003) Principles of Chemical Vapor Deposition ISBN-10: 1402012489;Mahan (2000) Physical Vapor Deposition of Thin Films ISBN-10:0471330019; Mattox (1998) Handbook of Physical Vapor Deposition (PVD)Processing (Materials Science and Process Technology Series) NoyesPublications ISBN-10: 0815514220; and Smith (1995) Thin-Film Deposition:Principles and Practice McGraw-Hill Professional ISBN-10: 0070585024.

In some aspects of the invention, microfabrication methods can be usedto produce islands of material within confined regions or nanoscaleapertures by first: providing a substrate having a cladding layer inwhich the cladding layer has an array of nanoscale apertures extendingthrough it to the substrate. Onto the cladding is deposited asacrificial layer. The sacrificial layer is generally deposited in aconformal manner to coat the walls of the apertures. After thesacrificial layer is deposited, the sacrificial layer is directionallyetched. The directional etching is carried out in a manner that resultsin the removal of the region of the sacrificial layer in the center ofthe nanoscale aperture, without the removal of the sacrificial layerfrom the walls. An island material is then deposited over the substratesuch that a portion of the island material becomes deposited in thecentral region of the aperture that was opened with the directionaletching. The island material is then etched such that a portion of theisland material within the aperture remains, and whereby portions of thesacrificial layer remaining are exposed. The sacrificial layer is thenremoved, leaving at least a portion of the island material within theaperture. This process produces an island of material within a nanoscaleaperture to which analyte molecules of interest can be attached.

FIG. 3 provides an exemplary method for using a sacrificial spacer toproduce a nanoscale apertures having islands disposed within them. Atransparent substrate 300 having a cladding layer on its surface isprovided. The cladding layer has an array of nanoscale apertures 315extending through the cladding 310 to the substrate 300. The substrate300 is generally a transparent substrate. In some cases, the transparentsubstrate comprises a silica based material such as fused silica (FuSi)or quartz. The cladding layer can be, for example a metal such asaluminum. In step (I) a first sacrificial layer 320 is deposited ontothe cladding layer. The deposition is carried out in a conformal mannerwhereby the walls of the aperture become coated with sacrificialmaterial. The sacrificial material can comprise, for example, silicon,germanium, or silicon/germanium. The first sacrificial layer can bedeposited, for example using chemical vapor deposition methods such asPECVD or LPCVD depending on the material deposited. In step (II) thesacrificial layer is etched in a directional manner. The directionaletching results generally in the removal of the sacrificial materialfrom the horizontal surfaces, leaving the sacrificial material coated onthe walls of the apertures. The deposition of the first sacrificial 320spacer is carried out in order to leave an opening with a crosssectional dimension of W_(O). The control of the size of this openingwill in turn control the size of the dimensions of the island. Thethickness of the first sacrificial spacer (T_(S)) on the walls of theaperture is related to the size of the opening by W_(O)=W_(Z)−2T_(S),where W_(Z) is the size of the opening of the aperture. The size of theisland, represented by W_(I), is on the order of W_(O), as thesacrificial layer acts as a mask during the etch step used to producethe island. In this step, it can be important to remove all of the firstsacrificial material from the region within the aperture in order toensure good adhesion of the island material.

In step (III) an island material 330 is deposited, for example in adirectional manner. A portion of the island material coats the exposedportions of the surface in the regions between the sacrificial layerscoating the walls of the apertures. The island material can comprise,for example, a metal such as gold, silver, platinum, nickel, or copper.The directional deposition of the island material can be carried out,for example using electron beam or thermal evaporation. In some cases, aseed layer, for example of titanium or chromium is used to improve theadhesion of the island material. A second sacrificial material 340 isdeposited in step (IV) in a manner whereby a portion of the secondsacrificial material is deposited on top of the portion of islandmaterial within the aperture. The second sacrificial material cancomprise silicon, germanium or silicon/germanium. In some embodiments,the first sacrificial layer and second sacrificial layer comprise thesame materials.

In step (V) an etching step is performed to remove the secondsacrificial layer and expose the portions of the island material that ison top of the cladding layer. The etching is carried out in a mannerwhereby the portion of the second sacrificial material within theaperture remains covering the portion of the island material within theaperture. In step (VI) an etching step is used to remove the portion ofthe island material over the cladding layer. Where the island materialcomprises gold, a wet etching step is typically employed. In this step,the remaining portion of second sacrificial material covers the islandmaterial within the aperture, preventing it from being etched andremoved. In step (VII) the first sacrificial layer and the secondsacrificial layer are removed. This removal is carried out in a mannerthat is selective to the cladding, the island material, and thesubstrate, such that these materials are not substantially removed inthe process. Removal of the sacrificial spacer layers can be carriedout, for example, using hydrogen peroxide or xenon difluoride. Thisprocess results in the formation of an island of material 360 within thenanoscale aperture. The island material is spaced within the apertureaway from the walls. Surrounding the island 360 is a region of substratesurface 350. The regions of substrate surface typically have differentenough chemical and physical properties to provide for the specificfunctionalization of the island material.

FIG. 4 provides an alternative method for microfabricating an island ofmaterial within an aperture which utilizes a gap-shirking approach usingan undercut. This method is amenable to high-throughput processing. Theundercut procedure provides a self-aligned approach to forming theisland. A dry undercut procedure can be employed to provide reliableundercut etching. In FIG. 4, first, a substrate 400 is provided havingthree layers on its top surface; first a layer of island material 410,then a layer of sacrificial material 420, followed by a layer of hardmask 440. The hard mask layer can be, for example, PECVD oxide ornitride, or any of the materials described herein for use as asacrificial layer. For this process, the hard mask layer must bedifferent than the sacrificial layer below it in order to allow forpreferential etching. In step (I), the hard mask layer is patterned andetched to form hard-mask structures 445 that will be used to define thenanoscale apertures. Next, in step (II) the sacrificial layer is etched,using the hard mask layer as a mask, to remove the portions of thesacrificial material not covered by the hard mask, and to undercut thehard mask to form pillars 425. The etching process can be carried out intwo steps, one to remove the bulk of the sacrificial material, and thesecond to perform the undercut, for example where the second step uses adry etch undercut.

In step (III), a cladding layer 450 is deposited. The cladding layer canbe a metal such as aluminum or an aluminum alloy. Step IV shows anoptional step in which the hard mask layer is removed. Where the hardmask is PECVD oxide, a Pad etch, for example, can be used. Pad etchsolutions are available from Ashland Chemical Company. Step (IV) isincluded, for example where it is desired to use an ion milling step in(V) where the presence of the hard mask could cause occlusion. In step(V), the island material layer is etched using the sacrificial pillarand the cladding layer as a mask. The island material can comprise ametal such as gold. The etch of the island material can be carried out,for example, using ion milling or with a wet etch. In step (VI) thesacrificial pillar is removed, for examples using hydrogen peroxide,XeF₂, or SF₆. Where step (IV) is not used, in some cases, the removal ofboth the pillar and the hard mask can be accomplished by etching awaythe sacrificial pillar. The process results in the formation of an arrayof apertures in the cladding layer, each aperture having an island 460of island material surrounded by exposed substrate 470.

In a number of embodiments of the invention, sacrificial layers aredeployed in a microfabrication process. The material that makes up thesacrificial layer depends on the manner that it is utilized and theother materials it is used with in the process. The suitable sacrificialmaterial will generally have the property whereby it can be selectivelyetched and removed without the etching and removal of other materialsexposed in that part of the process such as the cladding, substrate,island material, etc. In some cases, the sacrificial material comprisesa semiconductor material such as silicon, germanium, orsilicon/germanium. It can be, for example, amorphous silicon, amorphoussilicon carbide, or poly-germanium. In other cases, it can compriseother semiconductors such as III-V semiconductors including Ga—As. Insome cases, the sacrificial layer can comprise a metal such as titaniumor tungsten. The sacrificial layer can also comprise a dielectricmaterial such as a metal oxide or nitride including silicon oxide,silicon nitride, or titanium nitride. The sacrificial layer is generallyan inorganic material, but in some cases, it can comprise an organicmaterial such as parylene, photoresist, or another organic polymer. Insome cases a sacrificial layer can comprise a spin-on-glass, which aregenerally not preferred due to high temperature curing requirements, butwhich could be used in some circumstances, for example where highercuring temperatures can be tolerated. The sacrificial layer can becoated in the manner suitable for the material and structure of thedesired deposition. Suitable methods include, for example chemical vapordeposition (CVD), atomic layer deposition (ALD), sputtering,evaporation, electroplating, electroless plating, molecular beam epitaxy(MBE), or spin coating. In some cases, such as where the sacrificiallayer acts to coat the walls of apertures to provide an opening for theformation of an island, the coating of the sacrificial layer isperformed conformally, but not in a manner that will produce aplanarized coating. In some cases, such as where a second sacrificiallayer is coated to protect an island material from being etched in asubsequent step, the sacrificial layer can be deposited so as to form aplanarized coating.

For the case when the nanodot is gold, its diameter and height in a ZMWor other reaction region can be modulated and its adhesion to thesubstrate, if no adhesion promoter is used, can be achieved by exposingthe array to germane (GeH₄), e.g., as in Adhikari et al. (2007) J. Appl.Phys. 102:94311-94316. Au catalyzes decomposition of germane by thereaction GeH₄--->Ge+2H₂ (Woodruff et al. (2007) Nano Lett. 7:1637-1642)resulting in a Ge nano-wire with a Au—Ge tip as shown in FIGS. 5A and5B. As shown in FIG. 5B, solid/liquid nanoparticle 510 is situated uponsolid flat substrate 520 in the presence of GeH₄ vapor 540, resulting inthe formation of solid Ge nano-wire 560 with a tip composed ofsolid/liquid nanoparticle 510. The diameter and length of the nano-wirecan be controlled by modulating process conditions and exposure time toGeH₄. Prior to exposing the array to GeH4, the substrate can be exposedto high temperatures (ca. 300 degrees C.) to form spheres on thesubstrate. Anchoring the nanodot, e.g., through a germanium nano-wire tothe substrate, is not generally required for this class of embodiments.

The ZMW or other reaction region array structure can be formed usingavailable fabrication methods, e.g., forming the reaction region arrayover the completed nanostructure array, such that the nanostructuresreside in a desired portion of each of the reaction regions (e.g., inthe bottom of ZMW holes in a ZMW array). An example process forproducing an array of ZMWs or other suitable reaction regions caninclude: (1) surface cleaning, with the cleaning process type and recipebeing based on, e.g., the effectiveness of the deresisting process usedat the end of the production of the nanostructure array, adhesionstrength of the nanodots to the substrate and accumulated adventitiouscontamination due to time between steps and any storage environment; (2)deposition of a ZMW (or other array feature) cladding metal such asaluminum; (3) spin coating and post-application bake of a positive-tone,chemically-amplified resist; (4) e-beam or other suitable lithography;(5) post-exposure bake; (6) photoresist development; (7) photoresistdescum; (8) etch of the cladding metal; and (9) deresisting and finalcleaning. For an example illustration of this process, see the flowdiagram and illustration shown in FIG. 5C. Image placement orregistration errors of current electron beam technology is sufficientlyto provide accurate patterning of the zero mode waveguide structuresover the nanodot array, e.g., over a 6-inch square area. See, e.g.,Saitou (2005) “E-Beam Mask Writers,” in Handbook of PhotomaskManufacturing Technology, edited by S. Rizvi, New York, Taylor andFrancis. For example, the VB300 Electron Beam Lithography System fromVistec can achieve less than a 10 nm error in patterning. Availablee-beam systems used for photomask fabrication such as those from NuflareTechnology and JEOL have comparable image placement areas over a 6-inchsquare area Eynon and Wu (2005) Photomask Fabrication Technology, NewYork, McGraw-Hill; International Technology Roadmap for Semiconductors,2007 Edition. The cladding metal lithography technique for patterningsteps, nanodot metal adhesion promoters, and substrate material can bevaried with still accomplishing the main objective of the invention.Additional details on example implementations that providenanostructures in array regions such as ZMWs are provided below.

Fabricating or Immobilizing Nanoparticles in Arrays

One feature of the invention is the ability to achieve efficient highdensity loading of single molecules of interest into analysis regions ofan array. One class of embodiments achieves higher levels of single (orother desired number) occupancy loading into arrays or reaction regionssuch as ZMWs by fabricating a nanoparticle deposited or fabricated inthe reaction region. The nanoparticle is small enough that only one (oranother desired number) analyte can bind to the particle. While thisapproach is particularly useful for loading single molecules of analyte,e.g., for single molecule reactions (e.g., SMS), it will be appreciatedthat a desired number of particles can be deposited or fabricated inselected reaction regions to achieve specific loading of any desiredspecific number of analytes.

The nanoparticle(s) optionally include(s) an easily functionalizedsurface to permit attachment of an analyte of interest. For example, theparticle(s) can comprise gold, which can be functionalized with standardthiol chemistries. Individual particles are small enough that only adesired number of analytes (e.g., one) can bind to the particle, due tosteric interactions of the analyte at the surface of the particle.

For example, immobilization of, e.g., metal nanoparticles can beperformed by the process shown in FIG. 6. Metal nanoparticles of sizesranging from 10-100 nm are suspended in a negative-tone photoresist andspun onto a fused silica, synthetic quartz, borosilicate, or a similarsubstrate. Using e-beam lithography, Deep Ultra-Violet (DUV)lithography, nanoimprint, or other available lithography process,pillars ranging from 50-200 nm in diameter are fabricated. A metalcladding film such as aluminum is deposited onto the structure. Thephotoresist is removed in a manner that leaves a single nanoparticle ineach newly-created hole (e.g., comprising a reaction region (e.g., aZMW). Biotin/avidin/polymerase can be tethered on the nanoparticle(e.g., Au—S—(CH₂)x(C₂H₄O)y-biotin). The nanoparticles are small enoughthat only one polymerase or other analyte of interest can fit on them inthe reaction region, effectively limiting the number of analytes in thereaction region. During subsequent analyte loading processes, theanalyte can be loaded into the reaction regions at relatively highconcentrations, effectively loading most or all of the particles with ananalyte molecule. Excess analyte is washed from the reaction region,resulting in a high percentage of the reaction regions acquiring asingle polymerase or other analyte.

Depositing a Small Binding Site Island in a ZMW Using DirectionalDeposition

In one example approach, methods, systems and compositions fordepositing a small island or dot at the bottom of an array feature(e.g., ZMW) or even simply on a flat substrate to create a heterogeneoussurface of phase determining features, e.g., for single moleculeattachment are provided. As above, the island/dot can be, e.g., ametallic, dielectric, or semiconductor material on which a polymerase orother analyte can be immobilized by means of a linker molecule such as abiotin-terminated poly(ethylene glycol)alkanethiol, as noted in moredetail above.

In one aspect, in order to make islands sufficiently small so that onlyone polymerase can bind to one island, a high aspect ratio structure isused in conjunction with nonspecific, directional deposition. This caninclude, but is not limited to, physical vapor deposition (PVD) such assputtering, e-beam evaporation, and thermal evaporation, or chemicalvapor deposition (CVD) such as low-pressure CVD, plasma-enhanced CVD, orhigh density plasma CVD. Similar approaches have been used forfabricating nanowires of similar length scales, demonstrating the basicfeasibility of this approach. As shown in FIG. 7A, a high aspect ratiopattern is created by adding a buffer film between a photoresist and acladding film. Alternatively, a bilayer resist can be used in place of asingle-layer resist/buffer layer to create the necessary dimensions.After patterning the ZMW hole or other array feature with the desiredaspect ratio, a film is deposited over the entire structure by PVD orCVD. By using suitable aspect ratios and deposition conditions, a“breadloaf” shaped structure forms above the film, creating a maskthrough which a small diameter restricts the area of deposition onto thesubstrate. The breadloaf-like structure forms isotropically around,e.g., a ZMW hole, naturally aligning the island in the center of the ZMWcavity. The resulting island surface can be functionalized with linkerto bind the desired enzyme, e.g., a polymerase.

This overall process can also be adopted to form small dots that aresized for a single polymerase on flat substrates. These substrates canbe used with single molecule analysis techniques that do not require aZMW structure, such as Total Internal Reflectance Fluorescence (TIRF). Aprocess flow for flat substrates is shown in FIG. 7B.

Depositing a Small Binding Site Island in a ZMW Using Self-AssembledMonolayers and Atomic Layer Deposition

In this embodiment, to make islands sufficiently small so that only onepolymerase can bind to one island, imperfectly-formed self-assembledmonolayers and atomic layer deposition (ALD) are used. Thesetechnologies are routinely applied to form gate dielectrics of similarsize for nanoscale electronics, demonstrating the feasibility of thismethod. As shown in FIG. 8A, the imperfect monolayer serves as a maskfor forming an island on the surface by ALD. This island surface can befunctionalized with linker to bind the desired enzyme, e.g., apolymerase. Unlike other types of deposition processes such assputtering, evaporation, and conventional chemical vapor deposition(including but not limited to low-pressure chemical vapor deposition,high-density plasma chemical vapor deposition, plasma-enhanced chemicalvapor deposition, etc.), ALD is sensitive to surface species, and filmsdo not typically form unless those surface species are present to reactwith the ALD precursor and oxidizer. By selecting suitable monolayersfor the cladding film and substrate, the ALD film grows only on thesubstrate. Deposition of materials that form imperfect monolayers suchas octadecyltrichlorosilane is repeatable and controllable so that verysmall (<30 nm diameter) openings can be created, yielding an effectivenano-mask for ALD. As above, this process can also be adopted to formsmall clusters sized for single polymerases on flat substrates. Asabove, these substrates can be used with single molecule techniques thatdo not require a ZMW structure such as TIRF. A process flow for flatsubstrates is shown in FIG. 8B.

Additional details regarding the production of imperfect monolayers canbe found, e.g., in Richter et al. Phys. Rev. E, 2000, 61, 607-615.Further details regarding ALD can be found in Chen, et al., Appl. Phys.Lett., 2004, 84, 4017-4019.

Depositing Small Binding Site Islands in a ZMW Using a Spacer Film Inthis embodiment, in order to make e.g., dots or islands, sufficientlysmall so that only one polymerase may be bound to one island or dot, astructure similar to that used for transistor spacer films is used as aself-aligning masking layer which controls both the location and size ofthe island or dot. This method uses a spacer film or sacrificial layeras also described above. As shown in FIG. 9A, a multi-film stack iscreated by exposing a positive-tone photoresist and etching throughthree layers. Alternatively, a positive-tone-like resist pattern can becreated using nanoimprint lithography. The spacer film is deposited overthe etched pattern by atomic layer deposition (ALD) or chemical vapordeposition (CVD). Using a directional etch, a spacer structure iscreated that forms isotropically around the ZMW hole, naturally aligninga space to deposit the island film in the center of the ZMW cavity. Theisland material is deposited by physical vapor deposition (PVD) or CVD.The entire stack is planarized to expose the buffer layer and spacermaterial so they can be removed by wet etching. A polish stop/wet etchbarrier shown as the green film in FIG. 9A is present to protect thecladding film during these last two steps. Further details regardingtechniques useful to this embodiment are found in Cerofolini, et al.,(2005) Microelectr. Eng., 81, 405.

This process can also be adopted to form small islands that are sizedfor a single polymerase on flat substrates as well, e.g., using TIRF. Aprocess flow for flat substrates is shown in FIG. 9B. In this case, ifthe resist is used alone or in conjunction with the buffer layer, thenlow-temperature ALD is suitable for depositing over the resiststructures. The techniques illustrated by both FIGS. 9A and 9B producean island surface that can be functionalized with a linker, e.g., thatbinds to polymerase.

Deposit Metal Nanoparticles in a ZMW Using Backside Exposure ofPhotoresist

Immobilization of nanoparticles, e.g., metal nanoparticles can also beachieved by the process shown in FIG. 10. After the ZMW is fabricatedusing current processes, nanoparticles of sizes ranging from 10-100 nmare suspended in a negative-tone photoresist and spun onto the ZMWstructure. The backside of the ZMW is exposed to radiation to cross-linkthe resist. The wavelength is chosen so that the illumination region isat the bottom of the ZMW hole. The un-crosslinked resist is removed asusual. The remaining photoresist is removed by ashing or other mannerthat leaves the nanoparticles in the ZMW hole.

Creating Particles in a Target Area by Annealing Smaller Nanoparticles

In one embodiment, monolayers of small nanoparticles close-packed on asurface can be annealed to coalesce and form a single, larger particle.The technique uses deposition of monolayers of small nanoparticles (e.g.1.5 nm diameter particles) in a desired portion of an array (e.g., atthe bottom of ZMWs of the array) followed by annealing the sample. Theparticles coalesce to form 1 or a few larger particles in the bottom ofthe ZMW, providing a limited number of binding sites for an analyte ofinterest. The size of the resultant particle is dependent on thecomposition of the nanoparticle monolayer, e.g., spacing, density,particle size, etc. These parameters can be adjusted so that the size ofthe particle only allows a single polymerase to fit on it.

Deposition of a Gold Particle in a ZMW Using Block Copolymer MicelleNanolithography

In one aspect, block copolymer micelle nanolithography is used toproduce spatially well-defined deposits of nanometer-sized gold (orother nanomaterial) deposits that can be functionalized as phasedetermining features for binding of single analyte molecules. In thisfabrication protocol, ZMWs (or other small array features) act aspre-structured guides for self-assembly of block copolymer micelles,generated at a size to match the ZMW (or other array feature) diameter.This results in one micelle per waveguide, and also results inpositioning of an e.g., gold dot or cluster in the center of thewaveguide. The clusters are stable and immobile, presenting suitablesubstrate sites for coupling to suitably tagged or derivatized moleculesof interest, e.g., via gold-based or other suitable chemistries asdescribed above. The small size of the dots/clusters (e.g., gold dots assmall as 2 nm in diameter can be produced) ensures single moleculeoccupancy of the analyte in each ZMW (or other array feature), asproteins and other molecules are typically larger than the minimum sizeof the gold dots, and will be sterically prevented from binding morethan one molecule of analyte per dot (e.g., T7 DNA polymerase has a ˜10nm diameter); thus, the binding site is sterically inaccessible to moreanalyte molecules after the first analyte has bound. This permitsfunctionalization of a ZMW or other array reaction region underconditions of excess analyte (e.g., excess polymerase), ensuring thateach ZMW or other array region harbors a single analyte molecule,followed by washing to remove unbound enzyme.

Additional details regarding functionalization schemes for gold dotsprepared by block copolymer micelle nanolithography for binding ofsingle proteins can be found, e.g., in Glass et al. (2003) “Blockcopolymer micelle nanolithography” Nanotechnology 14:1153-1160; Glass etal. (2003) “Micro-nanostructured interfaces fabricated by the use ofinorganic block copolymer micellar monolayers as negative resist forelectron-beam lithography” Adv. Funct. Mat. 13: 569-575; Haupt et al.(2003) “Nanoporous gold films created using templates formed fromself-assembled structures of inorganic-block copolymer micelles” Adv.Mater. 15: 829-831; and Arnold et al. (2004) “Activation of integrinfunction by nanopatterned adhesive interfaces” Chemphyschem. 5:383-388.

Electrochemical Growth of a Nanostructured Polymerase Binding Site

In one class of embodiments, an electrical current is used to nucleategrowth of a nanostructure that can be used to bind to an analyte ofinterest. In this embodiment, an electrode can be placed under the ZMWor other array, with a transparent conductive substrate in between theelectrode and ZMW. A small amount of current flowing from the electrodenucleates the growth of a small nanostructure at the bottom of the ZMW.Once one such structure nucleates, it is far more likely for thatstructure to continue to grow in response to further current flow thanfor another nanostructure to nucleate. The current flow is turned off(stopping growth of the structure) while the structure is still smallenough for only one polymerase or other analyte to fit on it. Thenanostructure can be functionalized with the appropriate chemistry asdescribed herein. When the polymerases or other nanostructure is loadedin at high concentration, only one polymerase can bind to thenanostructure within each ZMW.

Depositing a Single Core-Shell Particle into a Single ZMW

As described herein, islands of particles can be created withinnanoscale apertures such as ZMW's by 1) Exposing the array of aperturesto core-shell particles which are sized such that one core-shellparticle is deposited into one aperture, then 2) removing the shellmaterial so as to deposit the core into the aperture. The core that isdeposited can be of a size such that only one molecule of interest suchas an enzyme will bind to it within the aperture, for example, with adiameter of from about 2 nm to about 20 nm. The core can be, forexample, a metal or metal oxide material. In some cases the core iscomprised of gold. The shell is generally comprised of a material thatcan be readily removed without adversely affecting the core particle orthe nanoscale aperture. In some cases, the shell comprises an organicpolymer. Organic polymer shells can be produced which result in gooddispersability in solution of the core-shell particle. The solution canbe brought into contact with an array of nanoscale apertures such thatgenerally one core-shell particle deposits in one nanoscale aperture.

After deposition, the organic material can be removed readily withoutdamaging the core particle or the nanoscale apertures. The organicmaterial can be selectively removed using a plasma, with hightemperature, and/or through oxidation. The plasma can be, for exampleeither an oxidizing or a reducing plasma. In some cases the treatmentfor removal of the shell also transforms the core material, for exampleeither through oxidation or reduction. The core particle can comprise,for example: Au, Pt, Pd, Ag, In, Fe, Zr, Al, Co, Ni, Ga, Sn, Zn, Ti, Siand Ge in the corresponding oxidation stages or mixtures thereof. Theshell material can comprise a polymer selected from block copolymers,graft copolymers, micro-branch polymers, star polymers having differentbranches, dendritic polymers, microgel particles, star block copolymers,block star polymers and core-shell latex polymers. In some cases, thepolymer is polystyrene-b-polyethylene oxide,polystyrene-b-poly(2-vinylpyridine), polystyrene-b-poly(4-vinylpyridine)or a mixture thereof. Methods for producing and depositing suchcore-shell polymers is described, for example, in Spatz et al., EuropeanPatent EP 1027157 and in Moller et al., European Patent EP 1244938.

The deposited core particle can be functionalized as described herein toattach a single molecule of interest to the particle. For example,selective methods of coupling to metals such as gold are known in theart and described herein. Thiol containing compounds can be used forselective coupling to a gold surface. For example, abiotin-PEG-alkane-thiol can be selectively reacted with the goldparticle. In some cases, in addition to coupling of a molecule ofinterest to the surface, it is useful to treat other surfaces in orderto prevent the unwanted binding of the molecule of interest or otherreaction compounds to other portions of the array.

In one exemplary embodiment, an array of nanoscale apertures in analuminum cladding on a silica surface are provided. The array is treatedwith silicon oxide by atomic layer deposition (ALD), resulting in asilicon oxide coating over the array. Micelles having metal or metaloxide salts such as HAuCl₄ in their cores and block copolymer shells aredeposited onto the array such that generally one micelle is deposited inone aperture. The array is treated with a plasma such that the blockcopolymer shell is degraded and removed and a single metal particle,e.g. a gold dot, having a diameter of about 8 nm to about 12 nm isdeposited in an aperture. The metal dot such as a gold dot isselectively functionalized with a coupling agent such asbiotin-PEG-silane-SH. The remaining portions of the surface are thenpassivated using silane chemistry, for example using treatment withsilane-PEG, where the PEG is terminated with methoxy, hydroxy, orcarboxylic acid. The molecule of interest, having a biotin attached toit is then coupled to the surface using avidin or streptavidin. The sizeof the functionalized metal dot and the size of the molecule ofinterest, such as an enzyme, are selected such that generally only onemolecule of interest is attached within each aperture.

Forming Single Analyte Binding Sites in an Array Feature

Similar to the approaches for putting a nanostructure into a ZMW (orother array feature) to facilitate super-Poisson loading of an analyte,the invention also provides approaches for locating binding groups thatare capable of binding to, e.g., a single analyte into the arrayfeature, without the use of a nanostructure to provide the binding site.In this class of embodiments, binding sites for analyte molecules arenot necessarily located on a functionalized particle or othernanostructure, but can be formed directly on the array feature. Forexample, using this class of embodiments, it is possible to form a smallfunctionalized region or island at the bottom of a ZMW that is capableof binding to an analyte molecule, e.g., where the functionalized regionis small enough that only one analyte molecule can bind to thefunctionalized region at a time.

As with the methods above, placement of a single analyte binding site inthe ZMW or other array reaction region makes it possible to completelyload the array with the desired number of analyte molecules (polymerase,template, etc.), by introducing a high concentration of analyte to thearray, and then washing any excess analyte from the array after bindingto the analyte binding site. As with the other embodiments herein, thisapproach is particularly well suited to delivery of a single analytemolecule to each reaction region of an array, though it is possible touse similar approaches to place more than 1 analyte binding site in anarray reaction region, if desired. For single molecule sequencingapplications, it is generally desirable to use the methods of this classof embodiments for loading single analyte molecules into a ZMW or otherarray reaction region.

In some embodiments the methods of microfabrication described herein canbe used to produce an island of functionality within a nanoscaleaperture such as an island of substrate material surrounded by a regionof isolation material, the substrate material and isolation materialshaving different enough chemical properties in order to selectivelyfunctionalize the substrate material for binding a single molecule ofinterest. Where the substrate surface comprises a silica based material,this material can be selectively functionalized using silane surfacechemistry. The silane chemistry can be used to functionalize a silicabased surface in the presence of an alumina surface. Methods forselective functionalization of surfaces are described in more detailherein.

FIG. 11 illustrates an embodiment of a method of the invention forforming an island of substrate within a nanoscale aperture surrounded byan isolation layer. A substrate 1100 having a cladding layer 1110 on topof it is provided. The cladding layer has an array of nanoscaleapertures 1105 extending through the cladding to the substrate. Thesubstrate is generally a transparent substrate such as fused silica orquartz. The cladding can be a metal such as aluminum or an alloy ofaluminum. In step (I) an isolation layer 1120 is deposited onto thesubstrate. The isolation layer is generally deposited as a conformallayer such that it coats the walls of the apertures as well as the base.The isolation layer is generally a relatively thin layer, for example,from about 2 nm to about 10 nm thick. The isolation layer can bedeposited using atomic layer deposition (ALD). The isolation layer cancomprise a metal oxide material such as aluminum oxide.

In step (II), a sacrificial material 1130 is conformally coated suchthat it coats the walls of the aperture. The thickness of thesacrificial layer is selected such that an opening between the coatedwalls remains. A lateral dimension of the opening, W_(O), is illustratedin FIG. 11. This opening will be utilized for the formation of theisland of functionality. The thickness of the sacrificial spacer (T_(S))on the walls of the aperture is related to the size of the opening byW_(O)=W_(Z)−2T_(S), where W_(Z) is the size of the opening of theaperture. The sacrificial material can comprise silicon, germanium orsilicon/germanium.

In step (III) the sacrificial layer is etched in a directional(anisotropic) manner to expose the surface of the isolation layer withinthe aperture, but to leave the sacrificial material on the walls of theaperture. In step (IV) the aluminum oxide isolation layer is etched.This etch can be a directional (anisotropic) etch. It can be carriedout, for example using Cl₂. In some cases, this etch can extend into thesubstrate.

In step (V) the sacrificial material is removed, for example usinghydrogen peroxide, XeF₂, or SF₆. The process produces an island 1150 ofsubstrate material, also referred to as a nanopit, within the nanoscaleaperture surrounded by a region of isolation material 1140. The size ofthe island, represented by W_(I), is on the order of W_(O), as thesacrificial layer acts as a mask during the etch step used to producethe island. In some cases in this embodiment and in other embodiments ofthe invention, the isolation layer will comprise alumina and thecladding will comprise aluminum metal. Where this is the case, thealuminum metal will generally have an aluminum oxide coating where ithas been exposed to air, thus all of the cladding surfaces will comprisealuminum oxide whether or not the isolation layer has been removed. Thisfeature can be useful where it is desired to subsequently treat, forexample to passivate, the surfaces that are not part of the island.Where substantially all of the surfaces outside of the island comprisealumina, one type of passivating chemistry can be used for all suchsurfaces, simplifying processing, thereby resulting in morereproducible, higher quality substrates.

While as shown, the cladding 1110 is directly deposited onto thesubstrate 1100, in some cases there can be a layer of material betweenthese layers. In some cases the additional layer will extend across thebase of the nanoscale aperture. Where this is the case, at the end ofthe process, the island within the aperture can have the surface of theintermediate layer rather than, or in addition to the surface of thesubstrate exposed.

FIG. 12 provides an alternative method for forming an island ofsubstrate surrounded by isolation layer within an array of nanoscaleapertures. A substrate 1200 is provided. The substrate is generally atransparent substrate, for example fused silica or quartz. In step (I)an isolation layer 1210 is deposited onto the transparent substrate. Theisolation layer is generally a relatively thin layer, for example, fromabout 2 nm to about 10 nm thick. The isolation layer can be depositedusing atomic layer deposition (ALD). The isolation layer can comprise ametal oxide material such as aluminum oxide.

In step (II), an array of nanopits 1220 is formed extending through theisolation material. These nanopits will become the islands of substrate,and thus are generally relatively small; having a lateral dimension frombetween about 2 nm and about 40 nm, or between about 5 nm and about 20nm. In some cases, due to the small size of the features, electron beamlithography or UV lithography can be used to produce the features. Whereelectron beam lithography is used, an e-beam resist such as ZEP520A, apositive tone resist from Zeon Corporation can be used. In other cases,high resolution lithography using UV can be used to define the nanopits.The etching of the alumina layer can be carried out using reactive ionetching (RIE), for example using a chlorine plasma. In some cases, theetching of the alumina layer results in some etching into thetransparent substrate. In some cases, it is desirable to etch into thetransparent substrate. The etching into the transparent substrate isgenerally fairly shallow, for example from about 1 nm to about 25 nm orabout 2 nm to about 10 nm into the substrate.

In step (III), a resist is used to produce a nanoscale feature or pillar1230 on top of each nanopit. The size and shape of the nanoscale feature1230 will determine the size and shape of the nanoscale aperture. Thenanoscale feature can be produced for example using a negative toneresist. Where electron beam lithography is used, a resist such as NEB31from Sumitomo Corporation can be used. In step (IV) a cladding layer1240 is deposited in a directional or line-of-sight manner, for examplethermally or with an electron gun. The deposition is carried out suchthat a gap remains at the edges of the nanoscale feature that is notcoated with cladding. Step (V) involves removing the nanoscale feature,thereby removing the cap of cladding above the nanoscale aperture andnanopit. Where the nanoscale feature 1230 is formed using a resist, asolvent such as 1165 remover from Shipley Corporation can be used. Theprocess produces a nanoscale aperture having an island of exposedsubstrate material 1260 surrounded by isolation material 1250. Where thecladding comprises aluminum, exposure of the cladding to aluminum willresult in the surfaces of the cladding comprising aluminum oxide. Wherethis is the case, and the isolation layer comprises aluminum oxide, oneset of passivation chemistry can be used to treat the surfaces outsideof the island of exposed substrate.

For the methods illustrated in FIG. 12, there are two separatelithography steps, one for producing the nanopits, and the other forproducing the nanoscale apertures. In order to ensure that the islandsend up near the center of the nanoscale apertures, it is important thatthere be accurate alignment between the two lithography steps. In orderto ensure good alignment, we have found that it is desirable to formregistration features in the substrate. The formation of registrationfeatures is generally carried out before carrying out the steps of FIG.12. In some cases the registration features are etched into thesubstrate. In other cases, the registration features are deposited ontothe substrate. FIG. 13A illustrates a preferred method for producingalignment features for this procedure. A substrate 1300 as describedabove is provided which has a hard mask layer 1310 such as aluminum ontop, and a resist 1320 on top of the hard mask. In step (I), the resistis developed and patterned. An electron beam resist such as ZEP520A fromZeon can be used for this step. In step (II) the hard mask is etched.Where the hard mask is aluminum, a chlorinated plasma reactive ion etchcan be used. In step (III) the substrate is etched. Where the substrateis fused silica, the substrate etch can be carried out using a fluorineplasma. The etch can be carried out to provide effective registrationmarks. The registration marks can be any appropriate shape, generallyhaving both vertical and horizontal lines when viewed from above. FIG.13(B) shows a representative substrate having a central region 1340comprising an array of nanoscale apertures each having an island ofexposed surface within them. The substrate has four registration marks1330, each in the shape of a “+”.

FIG. 14 shows an alternative method for producing an array of nanoscaleapertures, each having an island of exposed surface within them. Themethod generally involves depositing a single particle in the aperture,using that particle to mask a portion of the aperture base while anisolation layer is coated onto it, then removing the particle to exposethe portion of the surface the particle was masking. A substrate 1400having an array of nanoscale apertures 1405 through a cladding layer1410 is provided. In some cases, prior the substrate is coated with athin layer, for example by atomic layer deposition, over the claddingand the transparent substrate. This layer can comprise, for example,aluminum oxide.

In steps (I) and (II), an island particle that is smaller than theaperture is deposited within the nanoscale aperture. In step (I) acore-shell particle is deposited such that generally only one core-shellparticle 1420 is deposited in a single aperture. For example, the sizeof the core-shell particle is large enough that once one particle is inan aperture, the core-shell particle sterically prevents the depositionof a second core-shell particle in the well. The core-shell particle canbe, for example, a metal or metal oxide core particle surrounded by anorganic polymeric shell. In step (II), the shell portion of the particleis removed, thereby depositing a single small particle 1425corresponding to the core of the core-shell particle into the aperture.The shell can be removed, for example by treatment with a plasma whichselectively degrades the organic material comprising the shell. In somecases the core particle is simply deposited into the aperture, in othercases, the core is transformed during the shell removal process, forexample reduced from a salt or oxide to a metal. While in FIG. 14 thedeposition of the single particle is performed using a core-shellparticle, it is understood that the deposition can be performed in otherways including those described herein using other sizing moietiesproviding for the deposition of a single island-forming particle.

In step (III), an isolation layer 1430 is deposited to coat the regionsof the substrate surrounding the particle. This deposition can becarried out in a conformal manner such that the walls of the apertureare coated as shown in FIG. 14. In other embodiments the isolation layeris deposited in a directional manner to selectively coat the lateralsurfaces, and in other embodiments, the deposition can be carried outsuch that the isolation layer selectively grows on the substratesurface. The particle acts as a mask to prevent the deposition of theisolation layer onto a central portion of the base of the aperture. Theisolation layer can comprise an inorganic or an organic material. Theisolation layer can comprise, for example a metal oxide such as aluminumoxide or silicon oxide. In some cases, the isolation layer can comprisealuminum oxide that is deposited using atomic layer deposition (ALD).Where the substrate is a silica based material such as fused silica andthe particle comprises a metal such as gold, an oxide such as aluminumoxide can be selectively deposited onto the substrate and/or thecladding without substantially depositing onto the metal particle. Asdescribe above, in some cases, the substrate has a layer such as an ALDlayer over the substrate prior to step (I). In these cases, thedeposition of an isolation layer uses a different material than thefirst coating layer. In some embodiments, for example, an aluminum oxideALD layer is coated over the substrate prior to deposition of theparticles, and a silane, such as PEG-silane is deposited in step (III)as an isolation layer that is masked by the particle.

In an exemplary process, a particle such as gold is deposited intoapertures in an aluminum cladding on a silica surface. A silanepassivation layer, for example a silane-polyethylene glycol is depositedonto the portions of the silica surface surrounding the particle inorder to act as the isolation layer. In another optional step, thecladding is also passivated using a phosphate or phosphonate compoundsuch as Albritect™ CP30 or polyvinyl phosphonic acid (PVPA). Suitablesilanes and phosphate containing compounds are described, for example,in U. S. Patent Application 2008/0032301, which is incorporated hereinby reference for all purposes. In another exemplary process, a coatingsuch as a silicon oxide (SiO_(X)) coating or an aluminum oxide coatingis applied over an array having apertures in an aluminum cladding on asilica surface. This coating provides a uniform surface chemistry overthe array. Particles such as gold particles are deposited such thatgenerally one particle per aperture. Where a silicon oxide coating wasdeposited, silane chemistry is then used to produce an isolation layeron the array. Fore example, a silane-PEG is deposited onto the surfacesnot masked by the particle. The silane-PEG that is used can be methylterminated, hydroxy terminated, or carboxyl terminated. Where analuminum oxide coating is used, phosphonate or phosphate chemistry canbe used to passivate the regions of the aluminum oxide coating notmasked by the particle.

Once the isolation layer is deposited, in step (IV) the particle isremoved to expose the portion of the surface which the particle wasmasking. The removal of the particle exposes an island 1450 on thesurface that has a different surface chemistry than that of theisolation layer 1440 surrounding it, allowing for selective coupling ofa desired molecule onto the island. Where the exposed island comprises asilica surface, silane chemistry can be used to selectivelyfunctionalize the island. For example, a biotin-PEG-silane will bindselectively to the island surface without substantially binding to anisolation layer comprising a PEG coated surface. Where the exposedisland comprises aluminum oxide, phosphonate chemistry can be used toselectively attach a coupling agent, for example using abiotin-PEG-phosphonate or a biotin-PEG-PVPA. The particle can beremoved, for example, by selectively etching or dissolving the particle.Where the particle comprises gold, for example, an I₂/KI etchant or aKCN based etchant can be used. The core can comprise other metals suchas platinum, nickel, or aluminum. Where the deposited particle comprisesaluminum, phosphate based etchants can be employed to remove theparticle. Where the deposited particle comprises nickel, a FeCl₃ etchantcan be used.

The size of the masking particle can be varied by controlling the sizeof the core in the core-shell particle. Control of size of the core andthe shell of core-shell particles is known in the art, for example byforming micelles having cores comprising metal salts. See, for exampleSpatz et al., European Patent EP 1027157 and in Moller et al., EuropeanPatent EP 1244938. For example, the relative amount of metal salt, suchas HAuCl₄ or Pt(OAc)₂ in the formulation can be used to control the sizeof the metal particle that is deposited. The size of the shell can becontrolled by controlling the composition and the solvent conditions,for example by controlling the molecular weight of block copolymers usedto form the micelles. In some cases, the masking particle has a diameterof from about 2 nm to about 40 nm. In some cases, the masking particlehas a diameter from about 4 nm to about 20 nm. In some cases, themasking particle has a diameter from about 5 nm to about 15 nm.

In some cases, after functionalizing the island, for example with afunctionalizing agent such as biotin-PEG-silane, any excessfunctionalizing agent that is bound to the cladding rather than to theisland can be removed with an agent that specifically removes thefunctionalizing agent from the cladding. Such agents can comprise acidiccompounds, in particular compounds comprising phosphate or phosphonategroups. In some cases, the agent for specifically removing functionalityfrom the cladding is a phosphonate end-capped polymer such as polyvinylphosphonic acid (PVPA), Albritect CP-30, Albritect CP-10, AlbritectCP-90, Aquarite ESL, or Aquarite EC4020 from Rhodia, Inc.

FIG. 15 illustrates another embodiment of a method of the invention forforming an island of substrate surrounded by an isolation layer withinan aperture in an array of apertures. A substrate 1500 having asacrificial layer 1510 and a hard mask layer 1520 is provided. In step(I), the hard mask layer is patterned and etched to produce an array offeatures 1525 which will be used to define an array of nanoscaleapertures. In step (III) the sacrificial layer is etched, using the hardmask layer as a mask, to remove the portions of the sacrificial materialnot covered by the hard mask, and to undercut the hard mask to formpillars 1540. The etching process can be carried out in two steps, oneto remove the bulk of the sacrificial material, and the second toperform the undercut, for example where the second step uses a dry etchundercut. In step (III), a layer of isolation material 1550 isdeposited. The isolation material is deposited such that it extendsunder the overhanging hard mask layer to the base of the pillar ofsacrificial layer 1540. The isolation material can be, for examplealuminum oxide, deposited using sputtering. In step (IV) a claddinglayer 1560 is deposited. The cladding layer can be a metal such asaluminum or an aluminum alloy. The cladding material is deposited suchthat it does not extend under the overhanging hard mask layer to createan empty space between edge of the cladding layer and the pillar. Theisolation material can be deposited using evaporation. In step (V), thesacrificial layer and hard mask layer are removed, resulting in theremoval of the portions of the isolation layer and the cladding layer onthe portions of hard mask. In some cases, etching of the sacrificiallayer is performed, for example, using hydrogen peroxide, XeF₂, or SF₆.The process results in the formation of a nanoscale aperture having anisland of substrate surface at its base surrounded by a region ofisolation material.

Chemically Polishing a ZMW or Other Array Feature to Leave a SmallAnalyte Binding Site

In one approach, chemical polishing is used to form an analyte bindingsite at the bottom of a ZMW or other array reaction region. FIG. 16shows an example schematic of the process. An initial array of reactionregions 1620 (e.g., ZMWs) or other array features is formed in claddingmaterial 1600 (e.g., Al) on substrate 1610 (e.g., a glass, quartz orsilicon substrate). The initial cladding is thicker than the finaldesired thickness of the cladding in a final array, and reaction regions1620 (e.g., holes in the cladding), etc., have a diameter smaller thanthe final desired array region (e.g., the holes are smaller than theZMWs that form a ZMW array).

The first step in the process, shown in FIG. 16 subpanel (b), is todeposit functionalizing material 1630 such as peg-silane (e.g.biotin-peg-silane) on the surface of substrate 1610, which is covered incladding material 1600 (additional details regarding suitable linkingchemistries is found herein). The functionalizing material deposits onthe aluminum as well as on the glass or silicon surface at the bottom ofholes 1620 in the cladding material, but is removed during chemicalpolishing.

The polish step is provided by an immersion of the functionalizedcladded substrate into a phosphonic acid bath at elevated temperature(e.g. polyvinyl phosphonic acid at 90° C.). The acid uniformly etches(e.g., aluminum) cladding 1600. The surface at the bottom of the holes,e.g., glass, fused silicon, quartz, or the like, is not susceptible tocorrosion in phosphonic acid. The resulting structure, shown in FIG. 16subpanel (c), consists of a phosphonate treated ZMW (or other feature)array with a functionalized center having a larger diameter as theoriginal hole through the cladding. By controlling initial claddingthickness, material, and hole-diameter, it is possible to yield a verysmall functionalized area in the center of the fused silica surface. Thesize of the functionalized region can be small enough that only a singleappropriately functionalized analyte can bind to the smallfunctionalized region. For example, if the analyte is on the order of10-15 nm in diameter, the functionalized region can be on the order of,or less than, e.g., about 10-25 nm or less.

Such a surface is an ideal platform for achieving super-Poisson loadingof a single active analyte (e.g., polymerase) in the bottom of anotherwise passivated ZMW. If the functionalized area is small enough,then only one, e.g., polymerase, is able to bind within each ZMW.

Deposition of a Small Analyte Binding Site in an Array Feature Such as aZMW by Evaporation

In one embodiment, an evaporation strategy is used to leave afunctionalized region in the center of a ZMW or other array feature. TheZMW holes of the array are filled with one drop each of a solvent (wateror other solvent appropriate to the functionalization chemistry)containing a low concentration of solute. The solute is a linkermolecule capable of binding both the surface and the analyte (e.g.,capable of binding to a DNA polymerase). As the drop evaporates from theoutside to the center, the linker is concentrated in the middle of theZMW or other array feature. Eventually the linker precipitates in thecenter of the ZMWs of the array. The linker can be chemically absorbedto the ZMW by heating, exposure to light, or any other appropriatelinker fixation method. By optimizing the concentration of linker andsolvent it is possible to form a very small region of the linker in theZMW or other feature, e.g., small enough that only a single molecule ofanalyte can bind to the region.

The polymerase or other analyte is deposited into the ZMW or other arrayfeature, e.g., by flowing the analyte onto the array. Free analyte iswashed away, leaving an analyte bound to the center of the ZMW.

Tilted Angle Evaporation

In a variant of the above method, tilted angle evaporation is used tomask off a portion of an array feature (e.g., to mask off a portion of abottom surface of a ZMW), leaving a small unmasked region that can befunctionalized for analyte binding. The resulting functionalized regioncan be small enough that only a single analyte can bind to the region.In this embodiment, evaporation of a coating is performed in the arrayfeature (e.g., in the ZMWs of the array). If the sample is tilted in anevaporator, a portion of the bottom of the ZMW is left uncovered by thecoating. If the sample is rotated during the evaporation (FIG. 17), itis possible to form an uncoated island in the center of the ZMW thatdoes not contain the coating (this approach typically leaves the centerof the bottom of the ZMW uncoated, which is desirable). A binding sitefor the analyte (e.g., polymerase) can then be added to the island. Thebinding site only attaches to the bottom of the ZMW or other arrayfeature in the region of the uncoated island, i.e., the coating on therest of the bottom blocks functionalization of the coated regions. Thebinding site can be small enough so that only a single polymerase orother analyte will easily bind to it.

Non-Random Loading

One preferred aspect of the invention includes the non-random (andnon-Poisson limited) delivery of nucleic acids, enzymes and otheranalytes into the wells or other reaction regions of an array. Ingeneral, the analytes (and/or array components) of the invention can beconfigured so that a single analyte (or other desired number ofanalytes) is delivered per region. This can be achieved in any ofseveral ways as described herein, including by coupling moieties to theanalytes to sterically and/or electrostatically prevent loading of morethan one analyte, or, e.g., by incorporating a single binding site forthe analyte into the array region, or, e.g., by iterative loading ofanalytes, or, e.g., by actively controlling loading of the analyte, or,e.g., by temporarily or permanently configuring features of the array tocontrol analyte loading. These and other procedures are discussed indetail herein. The methods herein permit substantially more completeloading of single molecule analytes into arrays than is typical forrandom loading approaches, in which single molecule distributions ofanalyte are produced by underloading the array as a whole. As has beennoted, random distribution of analytes into the array results in one orfewer analytes being loaded into most reaction/observation volumes onlywhen fewer than about 36% of all observation volumes are loaded. Thistype of Poisson-limited analyte loading results in few enough moleculesbeing added to the array so that a Poisson-style random statisticaldistribution of the analyte molecules into the array results in one orfewer analytes per observation volume (in most cases). Prior art yieldsfor single-molecule occupancies of approximately 30% have been obtainedfor a range of ZMW diameters (e.g., 70-100 nm). See, Foquet (2008),herein. About 60% of the ZMWs in a ZMW array are not loaded (i.e., haveno analyte molecules) using such random loading methods.

The various methods of the invention can provide a frequency of as highas 100% loading for the relevant analyte of interest. Such high loadingefficiencies are possible, e.g., because the array does not typicallyaccept and/or bind more than one analyte in an analysis region of thearray (e.g., by distributing or fabricating one analyte binding particleper well, or one particle per analysis region of a well), or becausedelivery of the analyte to the well or other array region is controlled.By extending the appropriate incubation times and/or increasing theconcentration of particles, more complete loading is achieved. One ofskill can, of course, choose to load fewer than 100% of the wells of thearray. Typical particle-based arrays of the invention can includegreater than 30%, usually greater than 37% (the approximate Poissonrandom loading limit to achieve maximal single analyte moleculeoccupancy), typically 38% or more, often as much as 50% or more, andpreferably as much as 60%, 70%, 80% or 90% or more of the wells of thearray being loaded with a single molecule in an analysis region of eachwell (or, alternately, simply wells having a single analyte molecule perwell). In some cases an analyte molecule that is immobilized comprises abiomolecule such as an enzyme which has an activity. Where an activebiomolecule such as an enzyme is used, it is generally desired that theenzyme be active so that it can carry out its natural function, such ascatalyzing a chemical reaction. The methods, substrates, and systems ofthe invention allow for not only achieving higher levels of singlemolecule loading within wells, but higher levels of loading of activesingle molecules. Thus, typically 38% or more, often as much as 50% ormore, and preferably as much as 60%, 70%, 80% or 90% or more of thewells of the array being loaded with a single active molecule in ananalysis region of each well. A wide variety of methods, systems andcompositions for achieving non-random loading of particles are describedherein.

The array feature to be loaded with analyte is dependent on theapplication at issue and the equipment available. Arrays can includefeatures such as wells, depressions, grooves, waveguides, zero modewaveguides, chambers, microfluidic channels, trenches, magnetizedregions, unmagnetized regions, etched structures, machined structures,masked or unmasked analysis regions, masks that permit access by theanalyte to any analysis regions, arrangements of particles or otheranalyte binding sites in the array, arrangements of binding sites,located, e.g., at least 50 nm apart in the array, configured to bindindividual analyte molecules, and many other features can be loaded withanalyte according to the methods herein. The features can be arranged toprovide a physical phase determining feature, e.g., a regular ordecipherable pattern of locations into which the analyte is to beloaded. For example, the analyte molecules can be loaded into ZMWs orother features that are located in the array with a regular or selectedspacing that places the features, e.g., at least 20 nm apart, at least30 nm apart, at least 40 nm apart, at least 50 nm apart, at least 60 nmapart, at least 70 nm apart, at least 80 nm apart, at least 90 nm apart;at least 100 nm apart; at least 150 nm apart, at least 200 nm apart, atleast 250 nm apart, at least 300 nm apart, at least 350 nm apart, atleast 400 nm apart, at least 450 nm apart, or 500 nm or further apart.It should be appreciated that spacing of array features in the arraycan, of course be further apart, if desired, though this may decreasethe density of the features of the array, which may reduce overallthroughput of systems that comprise the array features. The phasedetermining feature can be simple location of the array features, e.g.,spacing of the array features on center in a regularly arranged physicalarray of features, or can be a more complex logically decipherablearrangement, e.g., where the features of the array are arranged in amanner that uses optical masking of signals from the array, and/or datadeconvolution algorithms to assign which features contribute to a“logical phase” of the array.

Particle and Other Sizing Moiety Regulated Delivery of Analytes toArrays

Particles or other sizing moieties can be selected such that a singleparticle/moiety fits into a single well/observation volume (e.g., ZMW)of a small well array. Sizing methods for sizing array wells to receivethe particles or moieties are discussed in more detail below; it isgenerally possible to control the size of wells to within a fewnanometers with respect to diameter and depth; ZMW arrays on the orderof 10 nm to greater than 200 nm can be achieved using available methods(see, e.g., Foquet et al (2008) “Improved fabrication of zero-modewaveguides for single-molecule detection” Journal of Applied Physics103: 034301; Eid et al. (2008) “Real-Time DNA Sequencing from SinglePolymerase Molecules” Science DOI: 10.1126/science.322.5905.1263b).Particle/moiety size is a function of the type of particle or othermoiety that is used for packaging or binding to the analyte of interest.

Analytes

A variety of analytes or molecules of interest can be delivered toreaction/observation regions using the methods and compositions herein.These include enzyme substrates, nucleic acid templates, primers, etc.,as well as polypeptides such as enzymes (e.g., polymerases).

A wide variety of nucleic acids can be analytes in the methods herein.These include cloned nucleic acids (DNA or RNA), expressed nucleicacids, genomic nucleic acids, amplified nucleic acids cDNAs, and thelike. Details regarding nucleic acids, including isolation, cloning andamplification can be found, e.g., in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al., MolecularCloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”); CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc; Kaufman et al. (2003) Handbook of Molecularand Cellular Methods in Biology and Medicine Second Edition Ceske (ed)CRC Press (Kaufman); and The Nucleic Acid Protocols Handbook RalphRapley (ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley).

Similarly, a wide variety of proteins, e.g., enzymes, can also bedelivered using the methods herein. The types of proteins can be bindingproteins that can be analyzed for binding with substrates in solution.These can comprise, for example receptor proteins and proteins that arepotential targets for drugs. A variety of protein isolation anddetection methods are known and can be used to isolate enzymes such aspolymerases, e.g., from recombinant cultures of cells expressing therecombinant polymerases of the invention. A variety of protein isolationand detection methods are well known in the art, including, e.g., thoseset forth in R. Scopes, Protein Purification, Springer-Verlag, N.Y.(1982); Deutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc. N.Y. (1990); Sandana (1997)Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996)Protein Methods, 2^(nd) Edition Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ, Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3^(rd) Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and thereferences cited therein. Additional details regarding proteinpurification and detection methods can be found in Satinder Ahuja ed.,Handbook of Bioseparations, Academic Press (2000). Sambrook, Ausubel,Kaufman, and Rapley supply additional useful details.

The enzyme can be any suitable enzyme that can operably be coupled to asurface. Examples include polymerases, DNA polymerases, RNA polymerases,reverse transcriptases, helicases, kinases, caspases, phosphatases,terminal transferases, endonucleases, exonucleases, dehydrogenases,proteases, beta-lactamases, beta-galactosidases, and luciferases.Examples of suitable enzymes include a Taq polymerase, an exonucleasedeficient Taq polymerase, an E. coli DNA Polymerase 1, a DNA polymeraseKlenow fragment, a reverse transcriptase, a wild type Φ29 polymerase, amutant Φ29 polymerase, an exonuclease deficient Φ29 polymerase, a T7 DNApolymerase, and a T5 DNA polymerase.

For a description of polymerases and other enzymes that are active whenbound to surfaces, which is useful in single molecule sequencingreactions in which the enzyme is fixed to a surface (e.g., to a particleor to a wall of a reaction/observation region, e.g., in a ZMW), e.g.,conducted in a ZMW, see Hanzel et al. ACTIVE SURFACE COUPLEDPOLYMERASES, WO 2007/075987 and Hanzel et al. PROTEIN ENGINEERINGSTRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS, WO2007/075873). For a description of polymerases that can incorporateappropriate labeled nucleotides, useful in the context of sequencing,see, e.g., Hanzel et al. POLYMERASES FOR NUCLEOTIDE ANALOGUEINCORPORATION, WO 2007/076057. For further descriptions of singlemolecule sequencing applications utilizing ZMWs, see Levene et al.(2003) “Zero Mode Waveguides for single Molecule Analysis at HighConcentrations,” Science 299:682-686; Eid et al. (2008) “Real-Time DNASequencing from Single Polymerase Molecules” Science DOI:10.1126/science.322.5905.1263b; Korlach et al. (2008) “Selectivealuminum passivation for targeted immobilization of single DNApolymerase molecules in zero-mode waveguide nanostructures” Proceedingsof the National Academy of Sciences U.S.A. 105(4): 1176-1181; Foquet etal. (2008) “Improved fabrication of zero-mode waveguides forsingle-molecule detection” Journal of Applied Physics 103, 034301;“Zero-Mode Waveguides for Single-Molecule Analysis at HighConcentrations” U.S. Pat. No. 7,033,764, U.S. Pat. No. 7,052,847, U.S.Pat. No. 7,056,661, and U.S. Pat. No. 7,056,676, the full disclosures ofwhich are incorporated herein by reference in their entirety for allpurposes.

Particles and Other Sizing Moieties

Sizing moieties such as particles can include either biological ornon-biological particle materials (or both). Thus, viral components areincluded within the definition of particles for purposes of the presentinvention as discussed above. In general, particles or other sizingmoieties to be delivered to the arrays of the invention can be formed ofany discrete material that can be coupled/associated, at leasttemporarily, to or with an analyte (e.g., a DNA, or an enzyme such as apolymerase) of interest, for delivery to the array of interest. Usefulparticles include a variety of polymer and ceramic beads,self-assembling structures such as nucleic acid origami (discussed inmore detail herein), as well as metal, glass, teflon, or silicaparticles. PEG or other large polymers can also be used to provide anappropriate particle/sizing moiety. For example, polymers, proteins,nucleic acids, polymer beads, silica beads, ceramic beads, glass beads,magnetic beads, metallic beads, and organic resin beads can be used toprovide particles in the context of the invention. The particles canhave essentially any shape, e.g., spherical, helical, spheroid, rodshaped, cone shaped, disk shaped, cubic, polyhedral or a combinationthereof. Optionally, they are configured to fit individually into therelevant well (e.g., ZMW) of the relevant array; the shape of therelevant particle can also be used to orient the particle in therelevant well, e.g., by shaping the walls of the well to conform to theparticle. Particles can optionally be coupled to any of a variety ofreagents that facilitate surface attachment of the analyte, e.g.,affinity matrix materials, or the like, e.g., nucleic acidsynthesis/coupling reagents, peptide synthesis/coupling reagents,polymer synthesis reagents, nucleic acids, nucleotides, nucleobases,nucleosides, peptides, amino acids, various monomers, biological samplematerials, synthetic molecules, or combinations thereof. In addition todelivering the analyte of interest, particles optionally serve a varietyof other purposes within the arrays of interest, including acting as“blank” or other control particles, calibration particles, sampledelivery particles, reagent particles, test particles, etc.

The particles as used herein typically comprise a core, for example ofisland material, and a shell. The whole particle including the core andthe shell is sized such that it can be delivered to the confined regionswith one particle being delivered to one region. This can beaccomplished, for example, by sizing the particle appropriately for fitinto the confined region. The conditions under which the particle isdelivered, such as the concentration of particles in solution, the modeof deposition, and the time and temperature of deposition, can also becontrolled such that a high percentage of confined regions only have oneparticle. Once the particles are delivered in a single particle toconfined region manner, the shell of the particle can be removed inorder to deposit the core material into the confined region. Forexample, the shell can comprise an organic material such as a polymerwhich can be dissolved or degraded from the core, depositing the corewithin the confined region. The conditions of the removal of the shellcan be controlled such that the core is deposited into a central regionwithin the confined region, producing an island of the core materialwithin the confined region, such as a well or ZMW. In some cases, theanalyte will be on the core prior to deposition. In other cases, theanalyte will be attached to the core after the core is deposited as anisland. For example, the core can be selected to form an island having asize such that generally only one analyte molecule or only one activeanalyte molecule will be attached to a single island due to stericconstraints.

Particles and other sizing moieties are sized to fit, optionallyindividually, into the array reaction/observation site (e.g.,reaction/observation portion of a ZMW or other well). Accordingly,particles will range in size, depending on the application, e.g., fromabout 1-500 nm in least one cross-sectional dimension. Typical sizes inZMW applications will range from about 5 nm to about 150 nm in at leastone dimension, e.g., about 25 to about 100 nm. In one useful embodiment,useful particles are about 50 nm to about 100 nm in at least onedimension. It is understood that a polymeric coating in solution willtend to extend into the solution, such that the outer boundary of theparticle is not a solid wall. In some cases the size of the particle canbe represented by its hydrodynamic radius.

The particles of the arrays of the invention can comprise essentiallyany material which can be moved into the array wells (e.g., ZMWs).Example particles include viral particles, as well as self-assemblingstructures, large nucleic acid or polypeptide complexes (including e.g.,ribosomes), polymeric, ceramic or metallic particles, beads, and thelike. For example, polymer beads (e.g., polystyrene, polypropylene,latex, nylon and many others), silica or silicon beads, ceramic beads,glass beads, magnetic beads, metallic beads and organic compound beadscan be used. An enormous variety of particles are commerciallyavailable, e.g., those typically used for chromatography (see, e.g.,Catalogs from Sigma-Aldrich (Saint Louis, Mo.), Supelco Analytical(Bellefonte, Pa.; sold, e.g., through Sigma-Aldrich), as well as thosecommonly used for affinity purification (e.g., the various magneticDynabeads™, which commonly include coupled reagents) supplied e.g., byInvitrogen. For a discussion of matrix materials see also, e.g., Hagelet al. (2007) Handbook of Process Chromatography, Second Edition:Development, Manufacturing, Validation and Economics, Academic Press;2nd edition ISBN-10: 0123740231; Miller (2004) Chromatography: Conceptsand Contrasts Wiley-Interscience; 2nd edition ISBN-10: 0471472077;Satinder Ahuja (2002) Chromatography and Separation Science (SST)(Separation Science and Technology Academic Press, ISBN-10: 0120449811;Weiss (1995) Ion Chromatography VCH Publishers Inc.; Baker (1995)Capillary Electrophoresis John Wiley and Sons; Marcel Dekker and Scott(1995) Techniques and Practices of Chromatography Marcel Dekker, Inc.

Delivering Particles to Arrays

Particles can be delivered to an array by methods that are generallyused to deliver analyte molecules to the array. For example, deliverymethods can include suspending the particles in a fluid and flowing theresulting suspension into the wells of the array. This can includesimply pipetting the relevant suspension onto one or more regions of thearray, or can include more active flow methods, such aselectro-direction or pressure-based fluid flow. In one usefulembodiment, the particles are flowed into selected regions of the array,e.g., where a particular particle type is to be analyzed in a particularregion of the array. This can be accomplished by masking techniques(applying a mask to direct fluid flow), or by active flow methods suchas electro-direction or pressure based fluid flow, including by ink-jetprinting methods. Ink jet and other delivery methods for deliveringnucleic acids and related reagents to arrays is found, e.g., in Kimmeland Oliver (Eds) 2006) DNA Microarrays Part A: Array Platforms &Wet-Bench Protocols, Volume 410 (Methods in Enzymology) ISBN-10:0121828158; Lee (2002) Microdrop Generation (Nano-and Microscience,Engineering, Technology and Medicine) CRC Press ISBN-10: 084931559X; andHeller (2002) “DNA MICROARRAY TECHNOLOGY: Devices, Systems, andApplications” Annual Review of Biomedical Engineering 4: 129-153.Microfluidic flow can also be used for analyte delivery; theseapproaches are discussed in more detail herein. Regions of an array canalso be selective targets of delivery simply by pipetting the relevantsuspension into the correct region of the array.

The arrays can incorporate or interface with fluid channels, e.g.,microchannels that can control or direct fluid flow into selectedregions of the array. Alternately, the fluid delivery methods can bediscrete from the array itself, e.g., using a print head, manualpipettor or robotic pipettor system. A variety of automated fluiddelivery systems are available and can readily be used in the context ofthe invention.

Iterative Analyte Loading

In one class of embodiments, high densities of single molecule analytesin array reaction regions such as ZMWs of a ZMW array are achievedthrough iterative loading procedures. In general, these proceduresinclude performing a first loading cycle, e.g., using standard randomanalyte loading methods, followed by a subsequent loading cycle thattargets regions of the array that were not loaded in the first loadingcycle. These iterative loading procedures can be repeated untilessentially complete single molecule loading into all desired arrayregions is achieved.

In general, after each loading cycle, the presence of the analyte ofinterest is detected, e.g., through an activity assay (e.g., bydetecting a SMS sequencing reaction), or via detection of a label boundto the analyte. Regions that do not comprise the analyte are targetedfor additional loading, e.g., by directing flow to those regions (e.g.,using microfluidic flow or optical or electrical trapping as describedherein), or simply by masking the loaded regions and loading unmaskedregions.

For example, random loading methods that result in a standardstatistical distribution of analytes into array regions can beperformed. For example, polymerase loading can be performed, using afluorescently labeled polymerase (or using any other type of detectablelabel). After deposition, the array is imaged (the labels are detected)to detect which array regions (e.g., which ZMWs) contain only onepolymerase (single versus multiple loading can be differentiated by themagnitude of the label signal at each array site). A mask is created,e.g., using lithographic methods as discussed herein, with the maskprotecting those array regions (e.g., ZMWs) that contain only onepolymerase. The remaining ZMWs are washed out to remove polymerases frommultiply-loaded ZMWs. This process can be repeated until essentially allof the array regions are loaded with analyte. For example, after tworounds, ˜60% of ZMWs can contain only one polymerase (if loadingproceeds using standard Poisson statistics, about 0.37+0.37*0.63). Afterthree rounds, ˜75% of ZMWs will contain only one polymerase. After fourrounds, ˜84%. After five rounds, ˜90%. After six rounds, ˜94% loading isachieved. If desired, the label can be cleaved from the polymerasebefore starting a sequencing or other reaction.

Alternatively, each step could utilize sub-Poisson loading (e.g., belowthe ˜37% Poisson limit) to ensure that there are virtually nomultiply-loaded ZMWs in each reaction cycle. In this case, it is notnecessary to differentiate between singly and doubly loaded arrayregions—instead, all of the labeled reaction sites can be masked, andthe loading process repeated on unlabeled sites.

Enrichment of Active Analytes

Typical analyte samples contain active and inactive forms of theanalyte. For example, a typical solution of polymerase enzyme containsmany copies of active and inactive molecules of polymerase. In bulksolution assays, where there are many copies of the analyte that can actin the reaction, this is not generally a significant issue—at most, itmay be useful to normalize the activity of the analyte for quantitativepurposes. However, in single molecule assays, the presence of a fractionof inactive molecules in a source of analyte molecules that is used toform single molecule reactions is undesirable, because the presence ofan inactive analyte molecule in a given single molecule reactioneffectively kills that reaction.

Thus, it is desirable to have a source of analytes that is enriched inactive analyte molecules. For example, it is useful to form singlemolecule sequencing reactions using an enriched population ofpolymerases that is, e.g., capable of DNA extension so that allpolymerase molecules immobilized in a SMS reaction are functional.Improperly active analytes such as some defective polymerases can alsohave undesirable features beyond simple inactivity, e.g., increasedbinding of labeled analogs, which can confound readout of SMS reactions;accordingly, it is also useful to actively eliminate improperly activeas well as inactive analytes.

In general, any of a variety of screening steps to negatively selectimproperly active analytes, combined with positive screening steps toisolate active enzymes can be used to achieve active analyte enrichment.For example, polymerases that bind, but do not release a template can benegatively selected, while polymerases that bind and extend a templatecan be selected for. Polymerases that do not bind template at all can benegatively selected. Centrifugation, or simple size or affinitypurification to isolate template bound from non-template bound fractionscan be used to purify active enzyme from inactive enzyme. Similarapproaches can be used to select for or against template nucleic acidsto be sequenced, e.g., by separating cross-linked from non-crosslinkednucleic acids, or the like.

In one example, the invention provides a protocol to enrich fraction ofactive polymerase (e.g., Phi29 polymerase) in a given sample. Thepolymerase sample is first incubated with a “double headed template”with a FAM or hapten conjugated at 5′ ends of oligos. Concentration ofpolymerase and template is to be at least about 10×Kd of thetemplate-Pol dissociation constant, with polymerase at slight excess toensure close to 100% binding of template. Functional polymerasemolecules bind template, but non-functional, inactive polymerase orcontaminating proteins do not. Bead conjugated to FAM antibody (oranother antibody that binds to the hapten) is mixed and centrifuged, andsupernatant is discarded to remove non-functional (non-template binding)proteins. The bead is resuspended and reagents for DNA extension areadded (divalent metal, dATP, dCTP, dGTP, dTTP) along with a trapmolecule, either DNA or heparin, at a concentration that is severaltimes in excess of the double-headed templates. The reaction is allowedto proceed for a few minutes to allow the polymerase to extend the DNA.Active, productive polymerase catalyzes dNTP incorporation and extendsthe DNA and eventually dissociates from the template when it reaches theend of the (linear) double headed template. These active polymerase bindthe trap molecule and do not rebind to any free “double head template”.Non-productive or non-catalyzing polymerase remains bound to thetemplate. Active, productive polymerase is separated by centrifugation,which pellets beads bound with nonproductive enzyme and template,leaving active, productive polymerase in the supernatant. Another methodto enrich active polymerase is to use magnetic beads conjugated to trapmolecules (DNA or heparin). The magnetic beads are added along withreagents for DNA extension. Active polymerase which dissociates from thetemplate binds to trap molecules on magnetic beads. Magnetic beads arethen separated from the reaction mixture. Active polymerase arerecovered by dissociating trap molecules, e.g., during dialysis.

An example flow chart for enrichment of active polymerases, e.g., usingphi29 as an example polymerase, is illustrated in FIG. 18. FIG. 19,panels A-B provides an example enrichment protocol. As illustrated,polymerase such as a phi29 polymerase can be enriched for the ability tobind to a template (linear, circular, etc.). Bound (active) and unbound(dead) polymerases are separated, e.g., by centrifugation. For example,the polymerase can be mixed with beads conjugated with receptor, e.g.,streptavidin for biotin, antibody for FAM, and incubated. The beads arethen pelleted and the supernatant, which contains unbound or inactivepolymerase, discarded. Polymerization is initiated by adding dNTPs anddivalent cation (Mg⁺⁺ or Mn⁺⁺) and active versus inactive polymerasesare again separated, e.g., using centrifugation. In another example,engaged polymerases are more stable at 37° C. than are non-engagedpolymerases, providing an additional enrichment selection scheme. A heattreatment before loading increases the proportion of productivepolymerase:template complexes, leading to improved loading. The additionof Ca²⁺ ions and cognate nucleotide analogs can be used to furtherimprove loading. Similarly, pre-forming and purifying astreptavidin-polymerase complex can be performed before template isadded to further enhance loading of active polymerase.

Further Details Regarding Linking Chemistries

The binding surfaces and/or particles within the arrays of the inventioncan present a solid or semi-solid surface for any of a variety ofavailable linking chemistries, allowing the binding of biologicalanalytes of interest to the particle members to be distributed into thearrays. A wide variety of organic and inorganic polymers, both naturaland synthetic can be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials that areemployed, include papers, ceramics such as glass, fused silicon, quartz,metals such as gold, metalloids, semiconductive materials, cements orthe like. In addition, substances that form matrixes, such as proteins(e.g., gelatins), lipopolysaccharides, silicates, agarose andpolyacrylamides can also be used. Proteins can also provide particles,e.g., using antibodies that bind specific recognition componentsincorporated into the analyte of interest.

A wide variety of linking chemistries are available for linkingmolecules to a wide variety of molecular, solid or semi-solid particlesupport elements. These chemistries can be performed in situ (i.e., inthe array) or prior to introduction of the particles into the array. Itis impractical and unnecessary to describe all of the possible knownlinking chemistries for linking molecules to a solid support. It isexpected that one of skill can easily select appropriate chemistries,depending on the intended application.

In one preferred embodiment, the particles or binding surfaces of theinvention comprise silicate elements (e.g., glass or silicate beads). Avariety of silicon-based molecules appropriate for functionalizingsurfaces are commercially available. See, for example, Silicon CompoundsRegistry and Review, United Chemical Technologies, Bristol, Pa.Additionally, the art in this area is very well developed and those ofskill will be able to choose an appropriate molecule for a givenpurpose. Appropriate molecules can be purchased commercially,synthesized de novo, or it can be formed by modifying an availablemolecule to produce one having the desired structure and/orcharacteristics.

A substrate linker attaches to the solid substrate through any of avariety of chemical bonds. For example, the linker is optionallyattached to the solid substrate using carbon-carbon bonds, for examplevia substrates having (poly)trifluorochloroethylene surfaces, orsiloxane bonds (using, for example, glass or silicon oxide as the solidsubstrate). Siloxane bonds with the surface of the substrate are formedin one embodiment via reactions of derivatization reagents bearingtrichlorosilyl or trialkoxysilyl groups. The particular linking group isselected based upon, e.g., its hydrophilic/hydrophobic properties wherepresentation of an attached polymer in solution is desirable. Groupswhich are suitable for attachment to a linking group include amine,hydroxyl, thiol (e.g., in the case of gold particles), carboxylic acid,ester, amide, isocyanate and isothiocyanate. Preferred derivatizinggroups include aminoalkyltrialkoxysilanes, hydroxyalkyltrialkoxysilanes,polyethyleneglycols, polyethyleneimine, polyacrylamide, polyvinylalcoholand combinations thereof.

By way of non-limiting example, the reactive groups on a number ofsiloxane functionalizing reagents can be converted to other usefulfunctional groups:

-   -   1. Hydroxyalkyl siloxanes (Silylate surface, functionalize with        diborane, and H2O2 to oxidize the alcohol);        -   a. allyl trichlorosilane→→3-hydroxypropyl        -   b. 7-oct-1-enyl trichlorchlorosilane→→8-hydroxyoctyl    -   2. Diol (dihydroxyalkyl) siloxanes (silylate surface and        hydrolyze to diol)        -   a. (glycidyl            trimethoxysilane→→(2,3-dihydroxypropyloxy)propyl    -   3. Aminoalkyl siloxanes (amines requiring no intermediate        functionalizing step)        -   a. 3-aminopropyl trimethoxysilane→aminopropyl    -   4. Dimeric secondary aminoalkyl siloxanes        -   a. bis (3-trimethoxysilylpropyl)            amine→bis(silyloxylpropyl)amine.

See, for example, Leyden et al., Symposium on Silylated Surfaces, Gordon& Breach 1980; Arkles, Chemtech 7, 766 (1977); and Plueddemann, SilaneCoupling Reagents, Plenum, N.Y., 1982. These examples are illustrativeand do not limit the types of reactive group interconversions which areuseful in conjunction with the present invention. Additional startingmaterials and reaction schemes will be apparent to those of skill in theart.

The components that can be attached to a derivatized particle or bindingsurface include nucleic acids such as DNA, polypeptides (e.g., enzymessuch as polymerases), mimetics, large and small organic molecules,polymers and combinations thereof. For example, moieties bearing acharge can be easily coupled to a particle. For example, the chargedgroup can be a carboxylate, quaternary amine or protonated amine that isa component of an amino acid that has a charged or potentially chargedside chain. The amino acids can be either those having a structure whichoccurs naturally or they can be of unnatural structure (i.e.,synthetic). Useful naturally occurring amino acids include: arginine,lysine, aspartic acid and glutamic acid. Surfaces utilizing acombination of these amino acids are also of use in the presentinvention. Further, peptides comprising one or more residues having acharged or potentially charged side chain are useful coating componentsand they can be synthesized utilizing arginine, lysine, aspartic acid,glutamic acid and combinations thereof. Useful unnatural amino acids arecommercially available or can be synthesized utilizing art-recognizedmethodologies, such as available systems of orthogonal elements. Inthose embodiments in which an amino acid moiety having an acidic orbasic side chain is used, these moieties can be attached to a surfacebearing a reactive group through standard peptide synthesismethodologies or easily accessible variations thereof. See, for example,Jones, Amino Acid and Peptide Synthesis, Oxford University Press,Oxford, 1992.

When proteins are attached to the particles or binding surfaces, it isalso possible to subsequently attach a nucleic acid to the protein. Forexample, a variety of proteins that specifically bind to specific DNAsequences can be used to link DNAs to the particles or binding surfaces.Examples include capsid packaging proteins, as discussed above, as wellas a variety of antibodies. Similarly, nucleic acids can be attached toparticles and used to bind polypeptides of interest. Linkers can beadded to the DNAs for purposes of linking to the proteins on theparticles or binding surfaces, using the methods discussed above, e.g.,in the context of adding packaging sites to the analyte nucleic acids.

Linking groups can also be placed on the particles of the invention.Linking groups of use in the present invention can have a range ofstructures, substituents and substitution patterns. They can, forexample be derivatized with nitrogen, oxygen and/or sulfur containinggroups which are pendent from, or integral to, the linker groupbackbone. Examples include, polyethers, polyacids (polyacrylic acid,polylactic acid), polyols (e.g., glycerol,), polyamines (e.g., spermine,spermidine) and molecules having more than one nitrogen, oxygen and/orsulfur moiety (e.g., 1,3-diamino-2-propanol, taurine). Specific examplesof linkers that can link DNA and proteins include: (1) incorporating06-benzylguanine analog(s) on DNA, and a SNAP-tag on the protein (Steinet al. (2007) “A Covalent Chemical Genotype-Phenotype Linkage for invitro Protein Evolution.” Chembiochem 8:2191-2194). Another known strongDNA-protein attachment that could be exploited is between the Tersequence at DNA replication terminators and the Tus protein, asdescribed by Coskun-Ari and Hill (1997) “Sequence-specific Interactionsin the Tus-Ter Complex and the Effect of Base Pair Substitutions onArrest of DNA Replication in Escherichia coli,” JBC 272:26448-26456.

In one embodiment of the invention, the coupling chemistries forcoupling materials to the particles of the invention arelight-controllable, i.e., utilize photo-reactive chemistries. The use ofphoto-reactive chemistries and masking strategies to activate couplingof molecules to substrates, as well as other photo-reactive chemistriesis generally known (e.g., for semi-conductor chip fabrication and forcoupling bio-polymers to solid phase materials). The use ofphoto-cleavable protecting groups and photo-masking permits typeswitching of particles, i.e., by altering the presence of substratespresent on the array members (i.e., in response to light). Among a widevariety of protecting groups which are useful are nitroveratryl(NVOC)-methylnitroveratryl (Menvoc), allyloxycarbonyl (ALLOC),fluorenylmethoxycarbonyl (FMOC), -methylnitro-piperonyloxycarbonyl(MeNPOC), —NH-FMOC groups, t-butyl esters, t-butyl ethers, and the like.Various exemplary protecting groups (including both photo-cleavable andnon-photo-cleavable groups) are described in, for example, Atherton etal., (1989) Solid Phase Peptide Synthesis, IRL Press, and Greene, et al.(1991) Protective Groups In Organic Chemistry, 2nd Ed., John Wiley &Sons, New York, N.Y. The use of these and other photo-cleavable linkinggroups for nucleic acid and peptide synthesis on solid supports is awell-established methodology.

Tethering Particles to the Array

The particles can incorporate features that permit tethering of theparticles to the wells of the array. Any of the applicable linkingchemistries discussed herein in the context of fixing analytes toparticles are applicable to the problem of linking/tethering theparticles to the surfaces of the arrays. Devices, methods and systemsthat incorporate functionalized regions into the walls of a ZMW, e.g.,by incorporating an annular gold ring into the walls of the ZMW, aredescribed, e.g., in Foquet et al. SUBSTRATES AND METHODS FOR SELECTIVEIMMOBILIZATION OF ACTIVE MOLECULES (U.S. Ser. No. 60/905,786, filed Mar.7, 2007 and U.S. Ser. No. 12/074,716, filed Mar. 5, 2008).

The particles can include appropriate functionalities for linking to therelevant array surface. For example, thiol chemistries can be used tolink proteins to surfaces. Recombinant proteins such as viral capsidassemblies can also include unnatural amino acids with any of a varietyof linking chemistries, e.g., when expressed in a host cell thatincludes orthogonal elements that permit site-specific expression of theunnatural amino acid. Systems of orthogonal elements that can be used toincorporate unnatural amino acids, including amino acids with reactivegroups, are described in Wang, et al. (2006) “Expanding the geneticcode.” Annu Rev Biophys Biomolec Struct 35: 225-249; Wang and Schultz(2005) “Expanding the Genetic Code,” Angewandte Chemie Int. Ed.44(1):34-66; Xie, et al. (2005) “An expanding genetic code.” Methods 36:227-38; and Xie, et al. (2006) “A chemical toolkit for proteins: anexpanded genetic code.” Nat Rev Mol Cell Biol 7: 775-82.

In the context of particles, the site specific incorporation of an aminoacid that comprises a reactive/linking group can be used to specificallyorient the particle relative to the array well. For example, the arraywell can include a specific functionalized region (e.g., a gold band, asdiscussed above) that can be coupled to a specific portion of theparticle. For example, where the particle is a viral particle, the tailor capsid can incorporate one or more reactive/linking groups to orientthe capsid relative to the well (and/or relative to other assaycomponents, such as surface immobilized enzymes, e.g., surfaceimmobilized polymerases).

Reading the Analyte

In the embodiments herein, the analyte molecule is optionally complexedto a particle, binding site or other entity and analyzed in a reactionsite, well, ZMW or other observation volume or region of the array. Inthe simplest case, this is accomplished simply by performing therelevant read reaction (e.g., a copy polymerization reaction using apolymerase); the analyte is optionally complexed to the particle, etc.,during this readout. This is particularly practical where the particleor other coupled moiety does not inhibit the action of relevant readoutcomponents, such as a polymerase analyte acting on a DNA templateanalyte. In the case of some viral particles, including manybacteriophage, the polymerase can capture the analyte DNA, which mayprotrude from the capsid, and can pull it from the capsid as itsynthesizes a complementary strand, e.g., during a sequencing reaction.Further, active enzymes can remain bound to particles, or can betransferred from a particle to a structure in the reaction/observationregion. See Hanzel et al. ACTIVE SURFACE COUPLED POLYMERASES, WO2007/075987 and Hanzel et al. PROTEIN ENGINEERING STRATEGIES TO OPTIMIZEACTIVITY OF SURFACE ATTACHED PROTEINS, WO 2007/075873). Similarly, apolymerase or other readout enzyme can bind to other particle-boundanalytes (e.g., enzyme substrates) and can act on them withoutseparation from the particle. However, alternate approaches can also beused, in which an analyte is separated from the particle or other moietybefore it participates in a relevant reaction.

Methods of separating the analyte from the particle are available in thecontext of the present invention. For example, a restriction enzyme canbe used to cleave an analyte DNA from the particle, after it isdelivered to an array well. Similarly, a polypeptide linker can becleaved using a site-specific protease. In another approach,photo-cleavable linkers can be used to couple the analyte to theparticle; upon exposure to light, the cleavable linker is cleaved,releasing the analyte. Linkers can also incorporate specificallycleavable linkages that cleave as a result of changing pH, presence of acleavage molecule, or the like. A viral capsid can be digested away fromthe nucleic acid using either chemical or enzymatic methods afterdelivery of the capsid to the array well. Any of these methods (orcombinations thereof) can result in a controllable release of theanalyte molecule from the particle of interest.

Once any necessary or desired separation of the analyte and anything itis bound to is performed, the analyte can be read or can participate inthe system in any of the typical methods that are used to read the arrayduring regular single molecule analyte monitoring. For example, in thecase of sequencing in a ZMW, a polymerase can be bound in the waveguidein which the sequencing reaction is performed; the incorporation ofappropriately labeled nucleotides is used to determine sequences of theanalyte nucleic acids. For a description of polymerases that canincorporate appropriate labeled nucleotides see, e.g., Hanzel et al.POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATION, WO 2007/076057. For adescription of polymerases that are active when bound to surfaces, whichis useful in single molecule sequencing reactions in which the enzyme isfixed to a surface, e.g., conducted in a zero mode waveguide, see Hanzelet al. ACTIVE SURFACE COUPLED POLYMERASES, WO 2007/075987 and Hanzel etal. PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OF SURFACEATTACHED PROTEINS, WO 2007/075873). For further descriptions of singlemolecule sequencing applications utilizing ZMWs, see Levene et al.(2003) “Zero Mode Waveguides for single Molecule Analysis at HighConcentrations,” Science 299:682-686; Eid et al. (2008) “Real-Time DNASequencing from Single Polymerase Molecules” Science DOI:10.1126/science.322.5905.1263b; U.S. Pat. No. 7,033,764, U.S. Pat. No.7,052,847, U.S. Pat. No. 7,056,661, and U.S. Pat. No. 7,056,676, thefull disclosures of which are incorporated herein by reference in theirentirety for all purposes.

In general, analytes such as nucleic acids or polypeptides can bedistributed into array wells using the methods described herein. Oncethe analytes are formatted into the appropriate wells, any of a varietyof different analyte readout formats in current use can be used duringanalyte analysis. These include fluorescence measurement,epifluorescence measurements, and the like. For a discussion of arrayreadout formats see, e.g., Kimmel and Oliver (Eds) (2006) DNAMicroarrays Part A: Array Platforms & Wet-Bench Protocols, Volume 410(Methods in Enzymology) ISBN-10: 0121828158; Kimmel and Oliver (Eds)(2006) DNA Microarrays, Part B: Databases and Statistics Volume 411(Methods in Enzymology) ISBN-10: 0121828166; Alan R. Kohane et al.(2005) Microarrays for an Integrative Genomics MIT Press ISBN:0262612100; Hardiman (2003) Microarrays Methods and Applications (Nuts &Bolts series) DNA Press, USA; Baldi and Hatfield (2002) DNA Microarraysand Gene Expression Cambridge University Press; ISBN: 0521800226;Bowtell and Sambrook (Eds) (2002) DNA Microarrays: A Molecular CloningManual David Paperback: 1st edition Cold Spring Harbor Laboratory; ISBN:0879696257; Microarrays and Related Technologies: Miniaturization andAcceleration of Genomics Research (May 1, 2001) Cambridge HealthtechInstitute ISBN: B00005TXRM; Rampal (ed) (2001) DNA Arrays: Methods andProtocols (Methods in Molecular Biology, Vol 170 Humana Press, ISBN:089603822X; Schena (2000) Microarray Biochip Technology Eaton Pub CoISBN: 1881299376; and Schena (Editor) (1999) DNA Microarrays: APractical Approach (Practical Approach Series) Oxford Univ Press, ISBN:0199637768. In general, a variety of commercially available arrayreaders exist, or can be modified to read the arrays of the invention.

EXAMPLES Example 1: Dimensional Values for Producing Nanoscale Islandsof Island Material

The methods illustrated in FIG. 4 are directed to producing islands ofnanodots within nanoscale apertures. FIG. 20A is a drawing illustratingthe values of relevant dimensions for the process illustrated in FIG. 4.Table 1 below shows values for the parameters for producing a goldnanodot on the order of 10 nm in diameter. The size of the gold dot canbe smaller than the diameter of the sacrificial pillar, due to undercutmade during gold wet etching.

TABLE 1 σ W(nano- W(ZMW) T(sacrificial) t(oxide) (undercut) T(gold) dot)1 100 nm 200 nm 50 nm 35 nm 10 nm ≦10 nm 2 120 nm 200 nm 50 nm 45 nm 10nm ≦10 nm 3 140 nm 200 nm 50 nm 55 nm 10 nm ≦10 nm

Example 2: Dimensional Values for Producing Nanoscale Islands ofSubstrate Surface

The methods illustrated in FIG. 15 are directed to the formation ofislands of substrate surrounded by an isolation layer within a nanoscaleaperture. FIG. 20B provides a drawing illustrating the values of thedimensions for the process illustrated in FIG. 15. Table 2 shows valuesfor the parameters for producing an island with a diameter on the orderof 10 nm.

TABLE 2 W(ZMW) T(sacrificial) t(oxide) σ (undercut) T(alumina) W(island)1 100 nm 200 nm 50 nm 45 nm 10 nm ≦10 nm 2 120 nm 200 nm 50 nm 55 nm 10nm ≦10 nm 3 140 nm 200 nm 50 nm 65 nm 10 nm ≦10 nm

Example 3: Gold Nano-Dot Islands

FIG. 21 shows Scanning Electron Micrographs of representative aperturesfrom an array of nanoscale apertures having gold dots disposed withinthem. The structures were formed using the method illustrated in FIG. 3.FIG. 21A shows a cross-section through a nanoscale aperture. FIG. 21Bshows an SEM taken from above the nanoscale aperture showing the goldnanodot at the base of the aperture. Starting from a ZMW having 100 nmthick aluminum on fused silica substrate, 30 nm PECVD amorphous siliconwas deposited at 300° C., followed by 7 nm gold/chrome evaporation atroom temperature and 9 μTorr. The gold/chrome at the top of ZMW could beremoved according to FIG. 3, or by tilted ion milling of gold. Finally,the sacrificial layer was removed in XeF2.

Example 4: Island of Exposed Substrate Surrounded by Isolation Layer

FIG. 22 shows a Transmission Electron Micrograph of a cross-section ofan aperture having an island of exposed substrate (fused silica)surrounded by isolation layer (aluminum oxide). The structure was formedby a process as illustrated in FIG. 12 and FIG. 13. Aluminum isdeposited onto a fused silica substrate. On top of the aluminum isdeposited a layer of e-beam resist ZEP520A. The resist is developed toproduce four cross-shaped registration marks on the substrate outside ofthe region in which the apertures will be formed. The width of the linesmaking up the alignment marks is about 5 microns. A chlorinated plasmareactive ion etch is used to etch the exposed portions of the aluminumlayer. A fluorine plasma is then used to etch the exposed portions ofthe fused silica using the aluminum as a hard mask. The etch extendsabout 1 micron into the fused silica substrate. The resist and aluminumare then removed. The aluminum is removed and the surface cleaned usinga Piranha etch.

Onto the substrate having the registration marks is deposited a layer ofaluminum oxide using atomic layer deposition (ALD) at thickness of about5 nm. An E-beam resist ZEP520A is then deposited onto the aluminum oxidelayer. The resist is patterned, developed, and the alumina layer etchedwith a chlorine RIE plasma to form nanopits of about 30-40 nm indiameter. The resist is removed using an oxygen plasma, and wetcleaning, using 1165 solvent. A negative resist NEB31 is deposited,patterned, and developed to form pillars which define the nanoscaleapertures on top of the nanopits. The pillars are aligned to the nanopitstructures using the registration marks. Aluminum is then depositedusing thermal or electron gun deposition. A solvent, for example 1165 isused to dissolve the resist pillars and lift-off portion of the claddingon the pillar to produce the structure shown in FIG. 22. It can be seenin FIG. 22 that the etching of the nanopit extends several nanometersinto the fused silica substrate.

Example 5: Island of Exposed Substrate Surrounded by Isolation Layer

FIGS. 23 and 24 show transmission electron micrographs of cross-sectionsof nanoscale apertures (ZMWs) having an island of fused silicasurrounded by an isolation layer of aluminum oxide. These structureswere formed by the process illustrated in FIG. 11. The cladding layer1110 is a layer of aluminum about 100 nm thick. The isolation layer 1120is a layer of aluminum oxide about 3.5 nm thick deposited by ALD at 300°C. The sacrificial layer 1130 is amorphous silicon at a thickness ofabout 30 nm which is deposited by ALD at 300° C. and etched using a Lam9600 etcher. The sacrificial layer is then removed using xenondifluoride which is selective in etching the sacrificial layer in thepresence of aluminum and fused silica. FIG. 24 is a close up view ofFIG. 23 which chose that Wi is around 26.5 nm and Wz is around 85 nm.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. For example, particle delivery canbe practiced with array well sizing methods as described. Allpublications, patents, patent applications, and/or other documents citedin this application are incorporated by reference in their entirety forall purposes to the same extent as if each individual publication,patent, patent application, and/or other document were individually andseparately indicated to be incorporated by reference for all purposes.

1. An array of nanoscale apertures comprising: a transparent substratehaving a cladding layer disposed on its surface, the cladding layerhaving a plurality of nanoscale apertures extending therethrough; eachnanoscale aperture having walls and a base, wherein an isolation layeris on the walls and on a portion of the base of the nanoscale aperture;wherein a portion of the base of the nanoscale aperture comprises anisland of substrate surrounded by isolation layer.
 2. The array of claim1 wherein the substrate comprises a silica based material.
 3. The arrayof claim 1 wherein the substrate comprises a fused silica.
 4. The arrayof claim 1 wherein the substrate comprises more than 10,000 nanoscaleapertures.
 5. The array of claim 1 wherein the nano scale aperturescomprise holes having a circular lateral profile.
 6. The array of claim1 wherein the apertures have a cross sectional dimension between 1 nmand 500 nm.
 7. The array of claim 1 wherein at least some of theplurality of nanoscale apertures comprise a single molecule of interest.8. The array of claim 7 wherein the single molecule of interestcomprises an enzyme.
 9. The array of claim 1 wherein at least some ofthe plurality of nanoscale apertures comprise a single active polymeraseenzyme, a single active template nucleic acid, or a single active primerattached to the island. 10-35. (canceled)
 36. A method for forming anisland of substrate surface within a nanoscale aperture comprising:providing a substrate having a cladding layer on top, the cladding layerhaving a plurality of nanoscale apertures extending therethrough;conformally depositing an isolation layer on the cladding layer andexposed portions of the substrate; conformally depositing a sacrificiallayer onto the top of the isolation layer; directionally etching thesacrificial layer such that the sacrificial layer remains on the wallsof the nanoscale apertures, and the sacrificial layer is removed fromthe region of the nanoscale aperture between the sacrificial layer onthe walls, exposing a portion of the isolation layer within thenanoscale aperture; etching the portion of the isolation layer withinthe nanoscale aperture to expose a portion of the substrate; andremoving the sacrificial layer to produce a structure having an islandof exposed substrate surface surrounded by isolation layer within eachnanoscale aperture.
 37. The method of claim 36 wherein the isolationlayer is deposited by atomic layer deposition (ALD).
 38. The method ofclaim 36 wherein the isolation layer comprises a metal oxide.
 39. Themethod of claim 38 wherein the isolation layer comprises alumina. 40.The method of claim 36 wherein the substrate comprises a transparentmaterial.
 41. The method of claim 40 wherein the substrate comprises asilica based material. 42-44. (canceled)
 45. The method of claim 36wherein the sacrificial layer comprises silicon or germanium or siliconand germanium.
 46. The method of claim 36 wherein the nanoscaleapertures comprise holes having a circular lateral profile.
 47. Themethod of claim 36 wherein the substrate comprises more than 10,000nanoscale apertures.
 48. The method of claim 36 further comprisingbinding a polymerase enzyme, template nucleic acid, or primerselectively onto the island.
 49. A method for forming an island ofsubstrate surface within a nanoscale aperture comprising: depositing anisolation layer onto a transparent substrate; forming nanopits in theisolation layer that extend to the substrate surface; depositing,exposing, and developing a resist to form a pillar of resist on top ofand extending over each nanopit; depositing a cladding layer such thatthe cladding layer covers the pillars of resist and the exposed regionsof isolation layer; and removing the resist resulting in lift-off of theportion of the cladding layer covering the pillars of resist, therebyforming a nanoscale apertures in the cladding layer, each having ananopit at its base surrounded by isolation layer. 50-94. (canceled)